2,2′-biphenazine compounds and methods useful for treating disease

ABSTRACT

The invention relates to a chemotherapeutic cancer treatment in which compounds (BH3Is) are administered to a mammal to treat B-cell Lymphoma or other hematopoietic cancers, including diseases associated with MCL-1. The invention also provides a method for treating types of hematopoietic cancers, such as B-cell lymphoma, using a combination of one or more disclosed compounds in combination with other therapies, for example, 26S proteosome inhibitors, such as, for example, Bortezomib. The invention also relates to autoimmune treatment with pharmaceutical compositions comprising one or more disclosed compounds. The invention also relates to methods for identifying compounds, for example, compounds of the BH3 mimic class, that have unique in vitro properties that predict in vivo efficacy against B-cell lymphoma tumors and other cancers as well as autoimmune disease. Illustrative compounds are those of Formula II:

RELATED APPLICATIONS

This application claims the benefit of Provisional Application61/425,909, filed Dec. 22, 2010, which is incorporated herein in itsentirety for all purposes.

GOVERNMENT SUPPORT

Research leading to this invention was in part funded by SBIR grantnumber R44 CA135915-02 from the National Cancer Institute, NationalInstitutes of Health, Bethesda, Md.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: a computer readableformat copy of the Sequence Listing (filename: EUTR_(—)006_(—)02USSeqList_ST25.txt.txt, date recorded: May 7, 2012, 2012, file size 1kilobyte).

FIELD OF THE INVENTION

This invention relates generally to compositions and methods fortreating cancer and autoimmune diseases. This cancer may behematological malignancies, such as Multiple Myeloma, B-cell lymphoma,and more specifically, the invention relates to treating autoimmunediseases and cancers, including hematological malignancies, with one ormore compounds that inhibit the Bcl-2 family of protein Mcl-1 as well asother of the Bcl-2 family proteins. In addition, this invention relatesto methods for determining selectivity of these newly classified “BH3mimic” compounds to predict efficacy in treating hematological and othermalignancies involving Mcl-1. All patents, patent applications, andpublications cited herein are hereby incorporated by reference in theirentireties.

BACKGROUND

Currently the prevalence of Multiple Myeloma (MM) is 63,000 people inthe US with about 13,000 new cases per year. There are 360,000 cases ofnon-Hodgkin's lymphoma (NHL) in the United States and 550,000 worldwide,with about 56,000 cases diagnosed per annum and 23,000 deaths per annum(American Cancer Society, The SEER Cancer Statistics Review (CSR) website, http://seer.cancer.gov/csr/1975_(—)2002/). Twenty percent of thesedo not respond to current therapy. In terms of all NHL cases, 60% areaggressive, of which 50% do not respond to front line therapy. Inaddition, chronic lymphocytic leukemia (CLL) is the most common form ofadult leukemia in the U.S. and in most of Western Europe. There areapproximately 70,000 cases of CLL in the U.S., with 10,000 new casesdiagnosed per annum (www.cancer.gov/cancertopics/types/leukemia). CLLpatients have a poor survival prognosis with a five-year survival rateof 46%.

Mcl-1 is a key regulator of lymphoid cancers including multiple myeloma(MM) (Zhang B et al. (2002), Blood 99: 1885-1893), non-Hodgkin'slymphomas (Cho-Vega J H et. al (2004) Hum. Pathol. 35(9): 1095-100) andchronic lymphocytic leukemia (CLL) (Michaels J, et al. (2004), Oncogene23: 4818-4827). Additionally, treatment of myeloma cells with theproteosome inhibitor Bortezomib (Velcade) has been shown to causeelevated Mcl-1 expression (Nencioni A, et al. (2005) Blood;105(8):3255-62) and this is seen in some myeloma patients (Podar K etal. (2008) Oncogene 27(6):721-31). It is proposed that an Mcl-1inhibitor would enhance the efficacy of Velcade treatment in MMpatients.

Though Rituxan, which targets B-cell surface protein CD-20, has provento be a valuable front line therapeutic for many NHL and CLL patients,resistance to this drug has been shown to correlate with elevatedexpression of B-cell lymphoma 2 (Bcl-2) or Myeloid Cell factor-1 (Mcl-1)proteins (Hanada et al. (1993) Blood 82: 1820-28; Bannerji et al. (2003)J. Clin. Oncol. 21(8): 1466-71). Notably, there is a high correlation ofelevated Mcl-1 with non-responsiveness to chemotherapies in B-cells fromCLL patients. (Kitada et al. (2002) Oncogene 21: 3459-74).Rituxan-resistant CLL cells also have a higher Mcl-1/Bax ratio thannormal cells, while there is no significant correlation of the Bcl-2/Baxratio. (Bannerji et al. (2003) supra).

Moreover, approximately 30% of diffuse large cell lymphomas (DLCLs) haveincreased Bcl-2 expression levels. This correlates with poor patientresponse to treatment with combination chemotherapy (Mounier et al.(2003) Blood 101: 4279-84). In follicular non-Hodgkin's lymphomas andplasma cell myeloma, Mcl-1 expression positively correlates withincreasing grade of the disease (Cho-Vega et al. (2004) Hum. Pathol.35(9): 1095-100).

The value of Bcl-2 as a target in anti-tumor therapy has been wellestablished. The literature also reports on Mcl-1 as a target intreating NHL, CLL, and acute mylogenous leukemia (AML) (Derenne et al.(2002) Blood, 100: 194-99; Kitada et al. (2004) J. Nat. Canc. Inst. 96:642-43; Petlickovski et al. (2005) Blood 105: 4820-28). Researchers haverecognized that proteins in the Bcl-2 family regulate apoptosis and arekey effectors of tumorigenesis (Reed (2002) Nat. Rev. Drug Discov. 1(2):111-21). Bcl-2 promotes cell survival and normal cell growth and isexpressed in many types of cells including lymphocytes, neurons andself-renewing cells, such as basal epithelial cells and hematopoieticprogenitor cells in the bone marrow.

In many cancers, anti-apoptotic Bcl-2 proteins, such as Bcl-2 and Mcl-1,unfortunately block the sensitivity of tumor cells to cytostatic orapoptosis inducing drugs. These proteins are therefore targets foranti-tumor therapy. A recently described class of small molecules thatinhibit Bcl-2 family proteins are the BH3 mimetic compounds (Nat.Reviews Drug Discovery vol 4: 399-409). These compounds function byinhibiting BH3 mediated protein/protein interactions among the Bcl-2family proteins. Several studies have described BH3 mimetic smallmolecules that function as Bcl-2 inhibitors by blocking BH3 binding(reviewed in Reed. et al. (2005) Blood 106: 408-418). Compounds with BH3mimic function include HA-14-1 (Wang et al. (2000) Proc. Natl. Acad.Sci. USA 97: 7124-9), Antimycin-A (Tzung et al. (2001) Nat. Cell. Biol.3: 183-191), BH3I-1 and BH3I-2 (Degterev et al. (2001) Nat. Cell. Biol.3: 173-82), and seven un-named compounds (Enyedy et al. (2001) J. MedChem 44: 4313-24), as well as a series of terphenyl derivatives (Kutzkiet al. (2002) J. Am. Chem. Soc. 124: 11838-9), and two new classes ofmolecules (Rosenberg et al. (2004) Anal. Biochem. (2004) 328: 131-8).More recently, a BH3 mimic compound has been tested in a mouse tumormodel (Oltersdorf et al. (2005) Nature 435(7042): 677-81).

The promise for using BH3 mimetic compounds as anti-tumor therapeuticshas been recognized, and is described in the literature; however, todate there are no conclusive reports from the clinic on the efficacy ofany anti-cancer drugs with this mode of action. While pharmacologicalmanipulation of the Bcl-2 family proteins is a feasible approach toachieving therapeutic benefit for cancer patients, the complexity of thenetwork of proteins that comprise this family makes this prospectdifficult. Therefore, with the large unmet medical need for treatinghematological malignancies, new approaches to assessing and utilizingthe detailed activity of the BH3 mimic molecules will have value indeveloping this class of therapeutics.

SUMMARY OF THE INVENTION

The present invention is directed to compositions and methods useful fortreating cancer and autoimmune diseases.

There is provided in accordance with one aspect of the invention,compounds of Formula Ia:Ar₁-L₁-Ar₂-L₂-Ar₃-L₃-Ar₄  Formula Iaand stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein:

Ar₁, Ar₂, Ar₃, and Ar₄ are each independently null, C₆₋₁₀ aryl, C₆₋₁₀heteroaryl, C₅₋₁₀ membered heterocycle, C₅-C₁₀ cycloalkyl, C₅-C₁₀heterocycle, C₁-C₆ straight or branched alkyl, each of which isoptionally substituted with one or more substituents R^(s);

L₁, L₂, and L₃ are each independently a covalent bond, or a saturated orunsaturated C₁₋₃ carbon chain, or a branched or unbranched C₁₋₃ carbonchain, wherein one or more methylene groups are optionally independentlyreplaced by heteroatoms chosen from O, NR and S(O)_(m); and wherein L isoptionally substituted with 0-2 oxo groups and one or more C₁₋₄ branchedor unbranched alkyl optionally substituted by one or more F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R,

each R is independently hydrogen, or linear or cyclic C₁₋₆ alkyl,branched or unbranched C₁₋₆ alkyl, or saturated or unsaturated C₁₋₆alkyl;

each m is independently 0, 1 or 2;

each R^(s) is independently F, Cl, Br, I, cyano, —C(O)R, C(O)NR₂,C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R, substituted or unsubstituted straightor branched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, or C₂₋₁₀ alkynyl, substituted orunsubstituted C₃₋₁₀ cycloalkyl, substituted or unsubstituted C₅₋₈cycloalkenyl, substituted or unsubstituted C₇₋₂₀ alkyl, substituted orunsubstituted saturated or unsaturated 3-11 member heterocyclyl orheterocyclylalkyl containing 1, 2, 3, or 4 heteroatoms selectedindependently from N, O, S(O)_(m);

provided that the compound of Formula Ia is not2,2′-(3,3′-dimethoxybiphenyl-4,4′-diyl)bis(azanediyl)dibenzoic acid orN4,N4′-bis(1-iminoisoindolin-5-yl)biphenyl-4,4′-diamine.

In another aspect, there is provided compounds of Formula Ib:

and stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein:

Ar₁ and Ar₄ are each independently null, C₆₋₁₀ aryl, C₆₋₁₀ heteroaryl,C₅₋₁₀ heterocycle, C₅-C₁₀ cycloalkyl, C₅-C₁₀ heterocycle, each of whichis optionally substituted with one or more substituents R¹, R², R³, R⁴,R⁵, R⁶, R⁷, and R⁸;

L₁ and L₃ are each independently a covalent bond, or a saturated orunsaturated C₁₋₃ carbon chain, or a branched or unbranched C₁₋₃ carbonchain, wherein one or more methylene groups are optionally independentlyreplaced by O, NR or S(O)_(m); and wherein L₁ and L₃ are optionallysubstituted with 0-2 oxo groups and one or more C₁₋₄ branched orunbranched alkyl optionally substituted by one or more F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R,

each R is independently hydrogen, or linear or cyclic C₁₋₆ alkyl,branched or unbranched C₁₋₆ alkyl, or saturated or unsaturated C₁₋₆alkyl;

each m is independently 0, 1 or 2;

each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is independently F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R, substitutedor unsubstituted straight or branched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, orC₂₋₁₀ alkynyl, substituted or unsubstituted C₃₋₁₀ cycloalkyl,substituted or unsubstituted C₅₋₈ cycloalkenyl, substituted orunsubstituted C₇₋₂₀ alkyl, substituted or unsubstituted saturated orunsaturated 3-11 member heterocyclyl or heterocyclylalkyl, containing 1,2, 3, or 4 heteroatoms selected independently from N, O, S(O)_(m);

provided that the compound of Formula Ib is not2,2′-(3,3′-dimethoxybiphenyl-4,4′-diyl)bis(azanediyl)dibenzoic acid, andAr₁ and Ar₄ are not simultaneously isoindolin-1-imine.

In another aspect, there is provided compounds of Formula Ic:

and stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein:

each X is independently selected from N or C to form a phenyl, pyridyl,pyrazinyl, pyrimidyl, triazinyl, or tetrazinyl moiety;

L₁ and L₃ are each independently a covalent bond, or a saturated orunsaturated C₁₋₃ carbon chain, or a branched or unbranched C₁₋₃ carbonchain, wherein one or more methylene groups are optionally independentlyreplaced by O, NR or S(O)_(m); and wherein L₁ and L₃ are optionallysubstituted with 0-2 oxo groups and one or more C₁₋₄ branched orunbranched alkyl optionally substituted by one or more F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R,

each R is independently hydrogen, or linear or cyclic C₁₋₆ alkyl,branched or unbranched C₁₋₆ alkyl, or saturated or unsaturated C₁₋₆alkyl;

each m is independently 0, 1 or 2;

each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, R¹⁷ and R¹⁸ is independently F, Cl, Br, I, cyano, —C(O)R, C(O)NR₂,C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R, substituted or unsubstituted straightor branched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, or C₂₋₁₀ alkynyl, substituted orunsubstituted C₃₋₁₀ cycloalkyl, substituted or unsubstituted C₅₋₈cycloalkenyl, substituted or unsubstituted C₇₋₂₀ alkyl, substituted orunsubstituted saturated or unsaturated 3-11 member heterocyclyl orheterocyclylalkyl containing 1, 2, 3, or 4 heteroatoms selectedindependently from N, O, S(O)_(m);

provided that the compound of Formula Ic is not2,2′-(3,3′-dimethoxybiphenyl-4,4′-diyl)bis(azanediyl)dibenzoic acid; and

wherein at least one of R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, andR¹⁸ is C(O)OR.

There is provided in accordance with another aspect of the invention,compounds of Formula II:

and stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein:

each of R¹, R³, R⁴, R⁶, R⁷, R⁸, and R⁹ is independently selected fromthe group consisting of hydrogen, hydroxyl, —(C₁-C₆) alkyl,—(C₁-C₆)alkylhydroxyl, —C₁-C₆)alkylamino, —(C₁-C₆)alkylamide,—O(C₁-C₆)alkylhalo, —OC(O)(C₁-C₆)alkyl, halo, —C(O)R, —C(O)NR₂, and—C(O)OR. Each R is independently selected from the groups consisting ofhydrogen, substituted or unsubstituted straight or branched C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, or C₂₋₁₀ alkynyl, substituted or unsubstituted C₃₋₁₀cycloalkyl, substituted or unsubstituted C₅₋₈ cycloalkenyl, substitutedor unsubstituted C₇₋₂₀ arylalkyl, substituted or unsubstituted saturatedor unsaturated 3-11 member heterocyclyl or heterocyclylalkyl containing1, 2, 3, or 4 heteroatoms selected independently from N, O, S.

There is provided in accordance with another aspect of the invention,pharmaceutical compositions comprising a compound of Formula Ia′:Ar₁-L₁-Ar₂-L₂-Ar₃-L₃-Ar₄  Formula Ia′or a stereoisomer, tautomer, solvate, or a pharmaceutically acceptablesalt, thereof, wherein:

Ar₁, Ar₂, Ar₃, and Ar₄ are each independently null, C₆₋₁₀ aryl, C₆₋₁₀heteroaryl, C₅₋₁₀ membered heterocycle, C₅-C₁₀ cycloalkyl, C₅-C₁₀heterocycle, C₁-C₆ straight or branched alkyl, each of which isoptionally substituted with one or more substituents R^(s);

L₁, L₂, and L₃ are each independently a covalent bond, or a saturated orunsaturated C₁₋₃ carbon chain, or a branched or unbranched C₁₋₃ carbonchain, wherein one or more methylene groups are optionally independentlyreplaced by heteroatoms chosen from O, NR and S(O)_(m); and wherein L isoptionally substituted with 0-2 oxo groups and one or more C₁₋₄ branchedor unbranched alkyl optionally substituted by one or more F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R,

each R is independently hydrogen, or linear or cyclic C₁₋₆ alkyl,branched or unbranched C₁₋₆ alkyl, or saturated or unsaturated C₁₋₆alkyl;

each m is independently 0, 1 or 2;

each R^(s) is independently F, Cl, Br, I, cyano, —C(O)R, C(O)NR₂,C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R, substituted or unsubstituted straightor branched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, or C₂₋₁₀ alkynyl, substituted orunsubstituted C₃₋₁₀ cycloalkyl, substituted or unsubstituted C₅₋₈cycloalkenyl, substituted or unsubstituted C₇₋₂₀ alkyl, substituted orunsubstituted saturated or unsaturated 3-11 member heterocyclyl orheterocyclylalkyl containing 1, 2, 3, or 4 heteroatoms selectedindependently from N, O, S(O)_(m); and a pharmaceutically acceptablecarrier.

There is provided in accordance with another aspect of the invention,pharmaceutical compositions comprising a compound of Formula Ib′:

or a stereoisomer, tautomer, solvate, or a pharmaceutically acceptablesalt, thereof, wherein:

Ar₁ and Ar₄ are each independently null, C₆₋₁₀ aryl, C₆₋₁₀ heteroaryl,C₅₋₁₀ heterocycle, C₅-C₁₀ cycloalkyl, C₅-C₁₀ heterocycle, each of whichis optionally substituted with one or more substituents R¹, R², R³, R⁴,R⁵, R⁶, R⁷, and R⁸;

L₁ and L₃ are each independently a covalent bond, or a saturated orunsaturated C₁₋₃ carbon chain, or a branched or unbranched C₁₋₃ carbonchain, wherein one or more methylene groups are optionally independentlyreplaced by O, NR or S(O)_(m); and wherein L₁ and L₃ are optionallysubstituted with 0-2 oxo groups and one or more C₁₋₄ branched orunbranched alkyl optionally substituted by one or more F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R,

each R is independently hydrogen, or linear or cyclic C₁₋₆ alkyl,branched or unbranched C₁₋₆ alkyl, or saturated or unsaturated C₁₋₆alkyl;

each m is independently 0, 1 or 2;

each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸ is independently F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R, substitutedor unsubstituted straight or branched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, orC₂₋₁₀ alkynyl, substituted or unsubstituted C₃₋₁₀ cycloalkyl,substituted or unsubstituted C₅₋₈ cycloalkenyl, substituted orunsubstituted C₇₋₂₀ alkyl, substituted or unsubstituted saturated orunsaturated 3-11 member heterocyclyl or heterocyclylalkyl, containing 1,2, 3, or 4 heteroatoms selected independently from N, O, S(O)_(m); and apharmaceutically acceptable carrier.

There is provided in accordance with another aspect of the invention,pharmaceutical compositions comprising a compound of Formula Ic′:

or a stereoisomer, tautomer, solvate, or a pharmaceutically acceptablesalt, thereof, wherein:

each X is independently selected from N or C to form a phenyl, pyridyl,pyrazinyl, pyrimidyl, triazinyl, or tetrazinyl moiety;

L₁ and L₃ are each independently a covalent bond, or a saturated orunsaturated C₁₋₃ carbon chain, or a branched or unbranched C₁₋₃ carbonchain, wherein one or more methylene groups are optionally independentlyreplaced by O, NR or S(O)_(m); and wherein L₁ and L₃ are optionallysubstituted with 0-2 oxo groups and one or more C₁₋₄ branched orunbranched alkyl optionally substituted by one or more F, Cl, Br, I,cyano, —C(O)R, C(O)NR₂, C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R,

each R is independently hydrogen, or linear or cyclic C₁₋₆ alkyl,branched or unbranched C₁₋₆ alkyl, or saturated or unsaturated C₁₋₆alkyl;

each m is independently 0, 1 or 2;

each R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵,R¹⁶, R¹⁷ and R¹⁸ is independently F, Cl, Br, I, cyano, —C(O)R, C(O)NR₂,C(O)OR, OR, NR₂, SiR₃, —S(O)_(m)R, substituted or unsubstituted straightor branched C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, or C₂₋₁₀ alkynyl, substituted orunsubstituted C₃₋₁₀ cycloalkyl, substituted or unsubstituted C₅₋₈cycloalkenyl, substituted or unsubstituted C₇₋₂₀ alkyl, substituted orunsubstituted saturated or unsaturated 3-11 member heterocyclyl orheterocyclylalkyl containing 1, 2, 3, or 4 heteroatoms selectedindependently from N, O, S(O)_(m); wherein at least one of R⁹, R¹⁰, R¹¹,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, and R¹⁸ is C(O)OR; and a pharmaceuticallyacceptable carrier.

There is provided in accordance with another aspect of the invention,pharmaceutical compositions comprising a compound of Formula II′:

or a stereoisomer, tautomer, solvate, or a pharmaceutically acceptablesalt, thereof, wherein:

each of R¹, R³, R⁴, R⁶, R⁷, R⁸, and R⁹ is independently selected fromthe group consisting of hydrogen, hydroxyl, —(C₁-C₆) alkyl,—(C₁-C₆)alkylhydroxyl, —(C₁-C₆)alkylamino, —(C₁-C₆)alkylamide,—O(C₁-C₆)alkylhalo, —OC(O)(C₁-C₆)alkyl, halo, —C(O)R, —C(O)NR₂, and—C(O)OR. Each R is independently selected from the groups consisting ofhydrogen, substituted or unsubstituted straight or branched C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, or C₂₋₁₀ alkynyl, substituted or unsubstituted C₃₋₁₀cycloalkyl, substituted or unsubstituted C₅₋₈ cycloalkenyl, substitutedor unsubstituted C₇₋₂₀ arylalkyl, substituted or unsubstituted saturatedor unsaturated 3-11 member heterocyclyl or heterocyclylalkyl containing1, 2, 3, or 4 heteroatoms selected independently from N, O, S; and apharmaceutically acceptable carrier.

In another aspect, the invention is directed to methods of treatingcancer in patients with pharmaceutical compositions comprising one ormore compounds of Formula Ia′, Ib′, Ic′, or II′. This cancer may be ahematological malignancies. Such hematological malignancies include, forexample, Multiple Myeloma, B-cell lymphoma, acute myelogenous leukemia,and chronic lymphocytic leukemia. Such treatment results in, forexample, tumor regression. Tumor regression can include, for example,killing a cancer cell.

In another aspect, the invention is directed to methods of treatingautoimmune diseases in patients with pharmaceutical compositionscomprising one or more compounds of Formula Ia′, Ib′, Ic′, or II′. Theautoimmune disease may be rheumatoid arthritis, osteo arthritis,psoriatic arthritis, psoriasis, neuromyaotonia, mayasthenia gravis,lupus erythematosus, endometriosis, Graves disease, granulomatosis,Crohns disease, interstitial cystitis, or multiple sclerosis.

A further embodiment of the invention is a method for treatingparticular types of hematopoietic cancers, using pharmaceuticalcompositions comprising one or more compounds of Formula Ia′, Ib′, Ic′,or II′. The use of these compounds for particular types of hematopoieticcancers have unexpected results in terms of efficacy and/or ability toinhibit particular anti-apoptotic (pro-survival) members of the Bcl-2family or to mimic particular members of the pro-apoptotic Bcl-2 familyproteins. Accordingly, hematological tumor cells that that arehyper-dependent on a particular member of the Bcl-2 family of proteinswill be most affected by that BH3 mimic which targets that protein.

Accordingly, in one aspect the invention provides a method for killing acancer cell comprising administering an amount of pharmaceuticalcompositions comprising one or more compounds of Formula Ia′, Ib′, Ic′,or II′ effective to kill a cancer cell of a hematopoietic cancer. Thetypes of hematopoietic cancer include, but are not limited to, MultipleMyeloma, B cell lymphoma, chronic lymphocytic leukemia, and acutemyelogenous leukemia.

In another aspect, the invention provides a method for killing a cancercell comprising administering an amount of pharmaceutical compositionscomprising one or more compounds of Formula Ia′, Ib′, Ic′, or II′ incombination with a 26S proteosome inhibitor to kill the cancer cell. Thetypes of 26S proteosome inhibitors include, but are not limited to,bortezomib.

In another aspect, the invention provides a method for killing a cancercell comprising administering an amount of pharmaceutical compositionscomprising one or more compounds of Formula Ia′, Ib′, Ic′, or II′ incombination with a chemotherapeutic agent that increases the level ofMcl-1 in the cancer cell. Such chemotherapeutic agents are 26Sproteosome inhibitors and inhibitors of the BH3 domain containing e3ligase called Mule. Such agents may be, but are not limited to,bortezomib or rituximab.

In another aspect, the invention provides a method for determiningwhether a candidate compound mimics a ligand specific for a target, themethod comprising the steps of (a) providing in a first reaction, thetarget, a first labeled peptide specific for the target, and a firstunlabeled peptide specific for the target, (b) providing in a secondreaction, the target, the first labeled peptide specific for the target,and a first candidate compound, (c) comparing binding specificity of thefirst unlabeled peptide with binding specificity of the first candidatecompound to determine whether the candidate compound mimics the firstunlabeled peptide. In certain embodiments, this method further comprisesrepeating steps (a), (b), and (c) wherein the first labeled peptidespecific for the target is replaced with a second labeled peptidespecific for the target. In certain embodiments, the target comprises aBH3 domain binding region, such as for example, a hydrophobic pocketformed by the BH1, BH2, BH3 and BH4 domains of the anti-apoptotic Bcl-2family of proteins.

In another aspect, the invention provides a method for treatingparticular types of hematopoietic cancers using a compound selected fromthe group consisting of pharmaceutical compositions comprising one ormore compounds of Formula Ia′, Ib′, Ic′, or II′. One or more of thesecompounds may inhibit the activity of the Bcl-2 family protein Mcl-1.

In one embodiment, the compounds of Formula Ia′, Ib′, Ic′, or II′ areused in a method for treating particular types of hematopoietic cancers,such as B-cell lymphoma, to preferentially inhibit the binding of apeptide comprised of the BH3 domain of Bak to the Bcl-2 family proteinMcl-1. This activity is unique among all of the reported BH3 mimics anddirects the use of this compound in treating certain hematologicalmalignancies that are affected principally by the Bcl-2 family proteinsand among those proteins, mostly by Mcl-1. Based on the unique abilityof compound A, or B or derivative, to inhibit BH3 binding to Mcl-1, thiscompound may be effective in blocking the unwanted cell survivalactivity of Mcl-1 in tumorogenic lymphoid and myeloid cells, andtherefore may be particularly effective as a therapy for treatingMultiple Myeloma (MM), diffuse large B-cell lymphoma (DLBCL), chroniclymphocytic leukemia (CLL), acute myelogenous leukemia (AML), all ofwhich are effected by elevated Mcl-1.

In another aspect, the invention provides a method for treatingparticular types of hematopoietic cancers using a combination of one ormore compounds selected from the group consisting of Formula Ia, Ib, Ic,or II, in combination with other therapies, for example, a class oftherapeutics known as 26S proteosome inhibitors, such as, for example,Bortezomib (Velcade®).

In another aspect, the invention provides methods for identifyingspecific activity of BH3 mimic compounds. For example, these compoundscan have varying potencies in inhibiting BH3-mediated binding ofparticular Bcl-2 family proteins, and the difference in potency can beidentified by systematically ordering combinations of protein-proteininteractions and comparing the blocking activity of BH3 mimic compoundsto that of competing BH3 domain containing peptides. By matching theactivity of the compound to a particular BH3 domain peptide, abiological activity can be assigned to that compound that correlates tothe activity of the BH3 domain containing protein. This information canbe used to predict the utility of a BH3 mimic compounds in treating aparticular disease.

In one aspect, the invention provides an agent, which modulatesapoptosis by binding to the Bcl-2 family proteins including Mcl-1 andpreferentially blocks BH3 domain binding.

In another aspect, the invention provides a method for usingpharmaceutical compositions comprising one or more compounds of FormulaIa′, Ib′, Ic′, or II′ as preferential inhibitors of Mcl-1.

In another aspect, the invention provides a method for blocking bindingof the BH3-only Bcl-2 family proteins or parts thereof, for examplePuma, Noxa, Bim, Bid and Bak, to Mcl-1.

In another aspect, the invention provides a method for usingpharmaceutical compositions comprising one or more compounds of FormulaIa′, Ib′, Ic′, or II′ as preferential inhibitors of Mcl-1 to induceapoptosis in cells overexpressing Mcl-1.

In another aspect, the invention provides a method for blocking bindingof the BH3-only proteins or parts thereof, for example Puma, Noxa, Bim,Bid and Bak, to Mcl-1.

In another aspect, the invention provides a method for using specificBH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′ for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatinglymphoid malignancies either alone or in combination with otheranti-tumor agents.

In another aspect, the invention provides a method for using specificBH3 mimic compounds or Formula Ia, Ib, Ic, or II for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatingmyeloid cancer either alone or in combination with other anti-tumoragents.

In another aspect, the invention provides a method for using specificBH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′ for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatingprostate cancer either alone or in combination with other anti-tumoragents.

In another aspect, the invention provides a method for using specificBH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′ for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatingnon-Hodgkin's lymphoma patients who are resistant to Rituxan eitheralone or in combination with other anti-tumor agents.

In another aspect, the invention provides a method for using specificBH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′ for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatingChronic Lymphocytic Leukemia patients who are resistant to Rituxaneither alone or in combination with other anti-tumor agents.

In another aspect, the invention provides a method for using specificBH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′ for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatingbreast cancer either alone or in combination with other anti-tumoragents.

In another aspect, the invention provides a method for using specificBH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′ for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatingliver cancer either alone or in combination with other anti-tumoragents.

In another aspect, the invention provides a method for using specificBH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′ for inhibiting theactivity of the Bcl-2 family protein Mcl-1 for the purpose of treatingovarian cancer either alone or in combination with other anti-tumoragents.

In another aspect, the invention provides a method for treatingmyelogenous leukemia patients with pharmaceutical compositionscomprising one or more compounds of Formula Ia′, Ib′, Ic′, or II′ incombination with Bortezomib or other proteosome inhibitors.

In another aspect, the invention provides a method for treating chroniclymphocytic leukemia patients with pharmaceutical compositionscomprising one or more compounds of Formula Ia′, Ib′, Ic′, or II′ incombination with Bortezomib.

In another aspect, the invention provides a method for treatingNon-Hodgkin's lymphoma patients with pharmaceutical compositionscomprising one or more compounds of Formula Ia′, Ib′, Ic′, or II′ incombination with Bortezomib.

In another aspect, the invention provides a method for treating breastcancer patients with pharmaceutical compositions comprising one or morecompounds of Formula Ia′, Ib′, Ic′, or II′ in combination withBortezomib.

In another aspect, the invention provides a method for treating prostatecancer patients with pharmaceutical compositions comprising one or morecompounds of Formula Ia′, Ib′, Ic′, or II′ in combination withBortezomib.

In another aspect, the invention provides a method for treating coloncancer patients with pharmaceutical compositions comprising one or morecompounds of Formula Ia′, Ib′, Ic′, or II′ in combination withBortezomib.

In another aspect, the invention provides a method for treatingpancreatic cancer patients with pharmaceutical compositions comprisingone or more compounds of Formula Ia′, Ib′, Ic′, or II′ in combinationwith Bortezomib.

In another aspect, the invention provides a method for treating livercancer patients with pharmaceutical compositions comprising one or morecompounds of Formula Ia′, Ib′, Ic′, or II′ in combination withBortezomib.

In another aspect, the invention provides a method for identifyingcompounds of the BH3 mimic class of small molecules that are activeagainst a subset of the BH3 domain containing proteins and thereforehave predicted efficacy against particular tumor types.

In another aspect, the invention provides a method of treating a mammalsuffering from migrating transformed B-Cell tumors (non-Hodgkin's)comprising the steps of administering pharmaceutical compositionscomprising a compound of Formula Ia′, Ib′, Ic′, or II′ and monitoringsaid mammal to determine state of said cancer; wherein said cancer is acancer sensitive to said chemical targeted to Bcl-2 family; optionallywherein the amount administered is a quantity sufficient to constituteeffective treatment, or wherein said cancer is chosen from a group ofcancers consisting of: lymphoma, breast cancer, leukemia, lung cancer,bone cancer, prostate cancer gastric cancer, colon cancer, rectalcancer, liver cancer, cervical cancer, renal cancer, bladder cancer,nasopharyngeal cancer, esophagus cancer, pituitary gland tumor; thyroidcancer melanoma and pancreatic cancer.

In another aspect, the invention provides a method of preventing cancercomprising the step of administering a chemical to persons having a highrisk of cancer.

In another aspect, the invention provides a method for selectingspecific activity of a BH3 mimic compound based on similar activity to apeptide comprised of a particular BH3 domain.

In various embodiments of the invention, a mammal is a human; cancer isMultiple Myeloma,

In various embodiments of the invention, a mammal is a human; cancer isNon-Hodgkin's Lymphoma; cancer is any other B-cell lymphomas; cancer isSmall lymphocytic, consistent CLL; cancer is Follicular, predominantlysmall cleaved cell; cancer is Follicular, mixed small cleaved and largecell; cancer is Intermediate grade Follicular, large cell; cancer isDiffuse, small cleaved cell; cancer is Diffuse, mixed small cleaved andlarge cell; cancer is Diffuse, large cell (cleaved and non-cleaved; saidcancer is High grade; cancer is Large cell, immunoblastic; cancer isLymphoblastic; cancer is Small non-cleaved cell; cancer is BurkittNon-Burkitt; cancer is Indolent NHL; cancer is B-cell CLL/smalllymphocytic lymphoma; cancer is Marginal zone lymphoma; cancer is MALT;cancer is Splenic marginal 27. 27; cancer is zone lymphoma; cancer isNodal marginal zone lymphoma; cancer is Lymphomplasmacytoidlymphoma/immunocytoma; cancer is Follicle center lymphoma, folliculartype Grade I (0-5 centroblasts/hpf) or Grade II (6-15 centroblasts/hpf)or Grade III† (>15 centroblasts/hpf); cancer is Aggressive NHL; canceris Diffuse, large cell lymphoma; cancer is Mediastinal large celllymphoma†; cancer is Primary effusion lymphoma; cancer is Mantle celllymphoma; cancer is Burkitt's lymphoma/high-grade Burkitt's-like; canceris Precursor B-cell leukemia/lymphoma; cancer is Precursor T-cellleukemia/lymphoma; cancer is skin cancer; cancer is prostate cancer;cancer is gastric cancer; cancer is colon cancer; cancer is rectalcancer; cancer is liver cancer; cancer is cervical cancer; cancer isrenal cancer; cancer is bladder cancer; cancer is nasopharyngeal cancer;cancer is esophagus cancer; cancer is pituitary gland tumor; cancer isthyroid cancer; cancer is melanoma; or cancer is pancreatic cancer.

In addition, in various embodiments of the invention, the chemical isadministered by injecting it directly into a tumor; the chemical isadministered by injecting it into said mammal's blood stream; thechemical is administered orally; the chemical is administered throughsaid mammal's skin; the chemical targeted to compounds of Formula Ia′,Ib′, Ic′, or II′ are administered in combination with prior artchemotherapy agents; or pharmaceutical compositions comprising acompound of Formula Ia′, Ib′, Ic′, or II′ are administered incombination with radiation therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The objects and features of the invention may be more fully understoodby reference to the drawings as described below in which:

FIG. 1 is a graph showing preferential competition of Bim-BH3 peptidebinding to Mcl-1 vs. Bim-BH3 peptide binding to Bcl-xL by the BH3 mimiccompound A. Fluorescence Polarization (FP) was used to measure bindingas described in Example 2.

FIG. 2 is a graph showing preferential competition of Bim-BH3 peptidebinding to Mcl-1 vs. Bim-BH3 peptide binding to Bcl-xL by the BH3 mimiccompound II-19. Fluorescence Polarization (FP) was used to measurebinding as described in Example 2.

FIG. 3 is a graph showing the efficacy of compound A, in killingmultiple myeloma-derived cells.

FIG. 4 is a set of graphs showing the efficacy of compound II-19, inkilling multiple myeloma derived cells (NCI-H929, FIG. 4 a), acutemyelogenousleukemia cells (AML-3, FIG. 4 b) and lymphoid derived cells(SUDHL-6, FIG. 4 c). On-target activity is indicated by the absence ofactivity against the Bax/Bak deficient cell line SUDHL-10 (FIG. 4 d).FIG. 4 e is a graph showing the efficacy of obatoclax in killingmultiple myeloma derivated cells (NCI-H929) and a Bax-Bak deficientlymphoid derived cell line (SUDHL-10).

FIG. 5 is a chart showing the ability of compound A to inducemitochondrial membrane permeabilization.

FIG. 6 is a chart demonstrating the selective activity of compound A ininducing mitochondrial membrane permeabilization in a manner that isdependent on Bax and Bak.

FIG. 7 is a chart showing the ability of compound II-19 to inducemitochondrial membrane permeabilization in an assay measuring therelease of cytochrome C. Response of mitochondria in semi-permeablizedlymphoid cell lines SUDHL-6 (right bars in each condition) and SUDHL-10(left bars in each condition) to compound II-19 demonstrating thatcompound II-19 causes release of cytochrome c in SUDHL-6BAX/BAK-functional cell line but not BAX/BAK-deficient SUDHL-10 cellline.

FIG. 8 is a graph summarizing the effect of compound A on white bloodcell count in a mouse human B cell leukemia mouse xenograft study.

FIG. 9 is a graph summarizing the activities of compound A in a mousehuman B cell leukemia (CCRF-CEM) mouse xenograft study.

FIG. 10 is a graph comparing the inhibition of Mcl-1 and Bcl-xL bycompound II-20 in the fluorescence polarization assay described inExample 2.

FIG. 11 is a graph of the pharmacokinetics of compound II-19 clearancein mouse plasma. Pharmacokinetic profile of Compound II-19 in male ICRmice is shown. Compound II-19 was dissolved in 5% DMSO, 15%hydroxypropyl-beta-cyclodextrain, and 50 mM Tris in water to yield anominal concentration of 5 mg/mL. This solution was administeredintraperintoneally to 24 male ICR mice at a dose of 10 mg/kg. Three micein each group were used for blood collection at each of 8 time points: 5minutes, 0.25 hrs, 0.5 hrs, 1 hr, 2 hrs, 4 hrs, 8 hrs, and 24 hrspostdose. Plasma samples at these time points were analyzed for thequantity of compound II-19 by standard liquid chromatography/massspectroscopy methods.

FIG. 12 is a graph showing the effect of compound II-19 on the growth oftumors derived from the myeloma cell line NCI H929 in a mouse humanmyeloma xenograft model. Activity of compound II-19 is compared tovehicle and to a dose of bortezomib known to show maximal activity.

The details of the invention are set forth in the accompanyingdescription below. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, illustrative methods and materials are now described.Other features, objects, and advantages of the invention will beapparent from the description and from the claims. In the specificationand the appended claims, the singular forms also include the pluralunless the context clearly dictates otherwise. Unless defined otherwise,all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term “heteroaryl” as used herein refers to a monocyclic or bicyclicring structure having 5 to 12 ring atoms wherein one or more of the ringatoms is a heteroatom, e.g. N, O or S and wherein one or more rings ofthe bicyclic ring structure is aromatic. Some examples of heteroaryl arepyridyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl,indolyl, tetrazolyl, benzofuryl, xanthenes and dihydroindole. It isunderstood that any of the substitutable hydrogens on a heteroaryl canbe substituted with halogen, C₁-C₃ alkyl, hydroxyl, alkoxy and cyanogroups.

As used herein “anti-apoptotic-protein” is a protein, which whenexpressed in a cell, blocks programmed cell death in cancer cells, orother cells that are otherwise being directed to undergo apoptosis.

As used herein “hematological malignancies” refers to any cancer of theblood or bone marrow, such as leukemia or lymphoma. Examples include butnot limited to: Myelomas (e.g. Multiple myeloma and Giant cell myeloma),Acute lymphoblastic leukemia (ALL), Acute myelogenous leukemia (AML),Acute myelogenous leukemia (AML), Chronic myelogenous leukemia (CML),Acute monocytic leukemia (AMOL), Hodgkin's lymphomas (all foursubtypes). In addition Non-Hodgkin's lymphomas such as Diffuse largeB-cell lymphoma (DLBCL),

Follicular lymphoma (FL), Mantle cell lymphoma (MCL), Marginal zonelymphoma (MZL), Burkitt's lymphoma (BL), Burkitt's lymphoma (BL), andother NK- or T-cell lymphomas are included.

As used herein, the term “Bcl-2” refers to the anti-apoptotic proteinoriginally discovered as the causal “oncogene” in lymphomas and firstdiscovered among the anti-apoptotic Bcl-2 family proteins.

As used herein “pro-apoptotic protein” means a protein that whenexpressed in a cell causes the cell to undergo apoptosis.

By “disrupts an interaction” is meant that a test compound decreases theability of two polypeptides to interact with each other. In certaininstances the disruption results in at least a 99% decrease in theability of the polypeptides to interact with each other. These wereidentified using a combination of virtual screening for molecularstructures, which fit the ideal structure of BH3 pocket and competitionbinding studies using fluorescence polarization (FP).

By “fluorescence polarization assay” is meant an assay in which aninteraction between two polypeptides is measured. In this assay, onepolypeptide is labeled with a fluorescent tag, and this polypeptideemits non-polarized light when excited with polarized light. Upon aninteraction of the tagged polypeptide with another polypeptide, thepolarization of emitted light is increased, and this increasedpolarization of light can be detected. By “interacts” is meant acompound that recognizes and binds to an anti-apoptotic protein butwhich does not substantially recognize and bind to other molecules in asample.

The present invention generally relates to compositions and methods fortreating hematological malignancies. Such hematological malignanciesinclude, for example, Multiple Myeloma, B-cell lymphoma, acutemyelogenous leukemia, and chronic lymphocytic leukemia. Such treatment,results in, for example, tumor regression in a mammal, such as a mouseor a human. Tumor regression can include, for example, killing a cancercell.

There is provided in accordance with one aspect of the invention,compounds of Formula Ia:Ar₁-L₁-Ar₂-L₂-Ar₃-L₃-Ar₄  Formula Iaand stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein

Ar₁, Ar₂, Ar₃, Ar₄, L₁, L₂ and L₃ are defined above for Formula Ia;

provided that the compound of Formula Ia is not2,2′-(3,3′-dimethoxybiphenyl-4,4′-diyl)bis(azanediyl)dibenzoic acid orN4,N4′-bis(1-iminoisoindolin-5-yl)biphenyl-4,4′-diamine.

In some embodiments, Ar₁, Ar₂, Ar₃, and Ar₄ are each independentlyselected from null, indazolyl, indolyl, isoindolyl, imidazolyl,benzimidazolyl, isoxazolyl, oxazolyl, thiazolyl, benzothiazolyl,piperidinyl, pyrazolyl, pyrazolinyl, pyrrolyl, pyrrolinyl, pyridinyl,pyridazinyl, pyrimidinyl, quinolinyl, isoquinolinyl, tetrahydroquinolyl,dihydroquinolyl, phthalazinyl, dihydroindolyl, indolinyl,benzoisoxazolyl, dihydrobenzoisoxazolyl, dihydroisoindolyl,benzoisothiazolyl, benzoisothiazolyl dioxide, cyclopentyl, cyclohexyl,tetrahydropyranyl, each of which is optionally substituted with one ormore substituents R^(s).

In some embodiments, Ar₁, Ar₂, Ar₃, and Ar₄ are each C₆-C₁₀ aryl.

In some embodiments, Ar₂ and Ar₃ are each C₆-C₁₀ aryl.

In some embodiments, Ar₂ and Ar₃ are each C₆ aryl.

In some embodiments, Ar₁ and Ar₄ are each C₆ aryl.

In some embodiments, Ar₁ and Ar₄ are the same.

In some embodiments, Ar₁ and Ar₄ are not the same.

In some embodiments, Ar₁ and Ar₄ are each null.

In some embodiments, Ar₁ and Ar₄ are each C₅₋₁₀ membered heterocyclic.

In some embodiments, Ar₁ is pyridinyl and Ar₄ is phenyl.

In some embodiments, Ar₁ and Ar₄ are each pyrimidinyl.

In some embodiments, Ar1 and Ar4 are each pyrazolyl.

In some embodiments, Ar1 and Ar4 are each isoxazolyl.

In some embodiments, Ar1 and Ar4 are each a quinolinyl.

In some embodiments, L2 is a bond.

In some embodiments, L2 is other than a bond.

In some embodiments, L2 is —S(O)₂—.

In some embodiments, L1 and L3 are the same.

In some embodiments, L1 and L3 are not the same.

In some embodiments, L1 and L3 are each —NR—.

In some embodiments, L1 and L3 are each —O—.

In some embodiments, L₁ is NR and L₃ is O.

In other illustrative embodiments, compounds of Formula Ia are set forthbelow:

-   2,2′-((Sulfonylbis(2-methoxy-4,1-phenylene))bis(azanediyl))dibenzoic    acid (Ia-1);

-   3,3′-((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dipropanoic    acid (Ia-2);

-   3,3′-((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(3-phenylpropanoic    acid) (Ia-3);

-   3,3′-((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(2-phenylpropanoic    acid) (Ia-4);

-   2,2′-((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(2-phenylacetic    acid) (Ia-5);

-   2,2′-(((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(sulfonyl))dibenzoic    acid (Ia-6);

-   2-((4′-((2-Carboxyphenyl)sulfonyl)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)sulfonyl)-6-methylbenzoic    acid (Ia-7);

-   Methyl    1-(2,2′,6,6′-tetrahydroxy-4′-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)-1H-indole-6-carboxylate    (Ia-8);

-   Methyl    2-oxo-1-(2,2′,6,6′-tetrahydroxy-4′-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)indoline-6-carboxylate    (Ia-9);

-   Methyl    2-oxo-3-(2,2′,6,6′-tetrahydroxy-4′-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)-2,3-dihydro-1H-benzo[d]imidazole-5-carboxylate    (Ia-10);

-   Methyl    2-oxo-1-(2,2′,6,6′-tetrahydroxy-4′-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)-1,2,3,4-tetrahydroquinoline-7-carboxylate    (Ia-11);

-   Methyl    1-(2,2′,6,6′-tetrahydroxy-4′-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)-1,2,3,4-tetrahydroquinoline-7-carboxylate    (Ia-12);

-   Methyl    2-oxo-1-(2,2′,6,6′-tetrahydroxy-4′-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)-1,2-dihydroquinoline-7-carboxylate    (Ia-13);

-   2-((2,2′,6,6′-Tetrahydroxy-4′-(7-(methoxycarbonyl)-2-oxo-3,4-dihydroquinolin-1(2H)-yl)-[1,1′-biphenyl]-4-yl)amino)benzoic    acid (Ia-14).

-   2-((4′-((2-carboxyphenyl)(methyl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)benzoic    acid (Ia-15);

-   2,2′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(methylazanediyl))dibenzoic    acid (Ia-16);

-   2-((4′-(2-carboxybenzyl)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)benzoic    acid (Ia-17);

-   2,2′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(methylene))dibenzoic    acid (Ia-18);

-   2,2′-((3,3′-dihydroxy-[1,1′-biphenyl]-4,4′-diyl)bis(oxy))dibenzoic    acid (Ia-19);

-   dimethyl    2,2′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dibenzoate    (Ia-20);

-   6,6′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(3-methylbenzoic    acid) (Ia-21).

In another aspect, there is provided compounds of Formula Ib:

and stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein

Ar₁, Ar₄ R¹, R², R³, R⁴, R⁵, R⁶, R⁷, and R⁸, L₁ and L₃ are as definedabove for Formula Ib, with the proviso that the compound of Formula Ibis not 2,2′-(3,3′-dimethoxybiphenyl-4,4′-diyl)bis(azanediyl)dibenzoicacid, and Ar₁ and Ar₄ are not simultaneously isoindolin-1-imine.

In some embodiments, Ar₁ and Ar₄ are each independently selected fromnull, indazolyl, indolyl, isoindolyl, imidazolyl, benzimidazolyl,isoxazolyl, oxazolyl, thiazolyl, benzothiazolyl, piperidinyl, pyrazolyl,pyrazolinyl, pyrrolyl, pyrrolinyl, pyridinyl, pyridazinyl, pyrimidinyl,quinolinyl, isoquinolinyl, tetrahydroquinolyl, dihydroquinolyl,phthalazinyl, dihydroindolyl, indolinyl, benzoisoxazolyl,dihydrobenzoisoxazolyl, dihydroisoindolyl, benzoisothiazolyl,benzoisothiazolyl dioxide, each of which is optionally substituted withone or more substituents R^(s).

In some embodiments, Ar₁ and A₄ are each independently selected fromcyclopentanyl, cyclohexyl, tetrahydropyranyl, and piperidinyl.

In some embodiments, Ar₁ and A₄ are each cyclopentanyl.

In some embodiments, Ar₁ and A₄ are each cyclohexyl.

In some embodiments, Ar₁ and A₄ are each piperidinyl.

In some embodiments, L₁ and L₃ are each NH.

In some embodiments, illustrative compounds of Formula Ib are describedbelow:

-   2,2′-((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dicyclopentanecarboxylic    acid (Ib-1);

-   2,2′-((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dicyclohexanecarboxylic    acid (Ib-2);

-   4,4′-((3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(tetrahydro-2H-pyran-3-carboxylic    acid) (Ib-3); and

-   1,1′-(3,3′-Dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(piperidine-2-carboxylic    acid) (Ib-4).

In another aspect, there is provided compounds of Formula Ic:

and stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein

Ar₁, and Ar₄ R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, L₁ and L₃ are as defined above for Formula Ic,with the proviso that the compound of Formula Ia is not2,2′-(3,3′-dimethoxybiphenyl-4,4′-diyl)bis(azanediyl)dibenzoic acid.

In other embodiment, illustrative compounds of Formula Ic are describedbelow:

-   3,3′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dibenzoic    acid (Ic-1);

-   2,2′-((3,3′-dihydroxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dibenzoic    acid (Ic-6);

-   2-((4′-((2-carboxyphenyl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)-5-methylbenzoic    acid (Ic-7);

-   3-((4′-((2-carboxyphenyl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)isonicotinic    acid (Ic-8);

-   6,6′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(3-methylbenzoic    acid) (Ic-17); and

-   2,2′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(4-methoxybenzoic    acid) (Ic-18);

-   2,2′-((((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(2,1-phenylene))bis(oxy))diacetic    acid (Ic-2);

-   2,2′-((2,2′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dibenzoic    acid (Ic-3);

-   2,2′-((2,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dibenzoic    acid (Ic-4);

-   2-((4′-(2-carboxyphenoxy)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)benzoic    acid (Ic-5);

-   5,5′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(pyrimidine-4-carboxylic    acid) (Ic-9);

-   4-((4′-((4-carboxy-1-methyl-1H-pyrazol-3-yl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)-1-methyl-1H-pyrazole-3-carboxylic    acid (Ic-10);

-   5-((4′-((5-carboxyisoxazol-4-yl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)isoxazole-4-carboxylic    acid (Ic-11);

-   5-((4′-((5-carboxy-2-methyloxazol-4-yl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)-2-methyloxazole-4-carboxylic    acid (Ic-12);

-   3,3′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))diisonicotinic    acid (Ic-13);

-   dimethyl    3,3′-((2-isopropoxy-2′-methoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(4-hydroxybenzoate)    (Ic-14);

-   dimethyl    3,3′-((2,2′,6,6′-tetrahydroxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(4-hydroxybenzoate)    (Ic-15);

-   dimethyl    3,3′-((2,2′-dihydroxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))bis(4-hydroxybenzoate)    (Ic-16);

-   2-((2,2′,6,6′-tetrahydroxy-4′-((2-hydroxy-5-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)amino)benzoic    acid (Ic-19);

-   2,2′-((2,2′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dibenzoic    acid (Ic-20).

Also described herein are compounds of Formula II:

and stereoisomers, tautomers, solvates, and pharmaceutically acceptablesalts, thereof, wherein

R¹, R³, R⁴, R⁶, R⁷, R⁸, and R⁹ are as defined above for Formula II.

In some embodiments, two of R¹, R³, R⁴, R⁶, R⁷, R⁸, and R⁹ are eachC(O)R.

In some embodiments, two of R¹, R³, R⁴, R⁶, R⁷, R⁸, and R⁹ are eachC(O)methyl.

In some embodiments, four of R¹, R³, R⁴, R⁶, R⁷, R⁸, and R⁹ are each—OH.

In some embodiments, each R⁹ is C(O)R.

In some embodiments, each R9 is C(O)methyl.

In some embodiments, each R1 and R6 is each OH.

In some embodiments, each R4 is C(O)R.

In some embodiments, each R4 is C(O)H.

In some embodiments, each R4 is CH2OH.

In other embodiments, illustrative compounds of Formula II are describedbelow:

-   dimethyl    4,4′-diformyl-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-19); and

-   dimethyl    1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-20).

-   dimethyl    1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-1);

-   1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylic acid    (II-2);

-   1,1′-(1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-diyl)diethanone    (II-3);

-   9,9′-bis(1-hydroxyethyl)-[2,2′-biphenazine]-1,1′,3,3′,6,6′-hexaol    (II-4);

-   9,9′-bis(2-hydroxypropan-2-yl)-[2,2′-biphenazine]-1,1′,3,3′,6,6′-hexaol    (II-5);

-   9,9′-diisopropyl-[2,2′-biphenazine]-1,1′,3,3′,6,6′-hexaol (II-6);

-   [2,2′-biphenazine]-1,1′,3,3′,6,6′-hexaol (II-7);

-   dimethyl    4,4′-bis((dimethylamino)methyl)-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-8);

-   dimethyl    4,4′-bis((benzylamino)methyl)-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-9);

-   dimethyl    1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis((methylamino)methyl)-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-10);

-   dimethyl    1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis((isopropylamino)methyl)-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-11);

-   dimethyl    1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis((phenylamino)methyl)-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-12);

-   dimethyl    1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(pyrrolidin-1-ylmethyl)-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-13);

-   dimethyl    4,4′-bis(((cyclopropylmethyl)amino)methyl)-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-14);

-   dimethyl    1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-15);

-   dimethyl    4,4′-bis(aminomethyl)-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-16);

-   dimethyl    4,4′-bis(acetamidomethyl)-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-17);

-   dimethyl    4,4′-bis(benzamidomethyl)-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate    (II-18);

-   N9,N9′-dibenzyl-1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-[2,2′-biphenazine]-9,9′-dicarboxamide    (II-21);

-   1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-N9,N9′-bis(2-phenylpropyl)-[2,2′-biphenazine]-9,9′-dicarboxamide    (II-22);

-   1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-N9,N9′-diisobutyl-[2,2′-biphenazine]-9,9′-dicarboxamide    (II-23);

-   1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-N9,N9′-diisopropyl-[2,2′-biphenazine]-9,9′-dicarboxamide    (II-24); and

-   1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-N9,N9′-dimethyl-[2,2′-biphenazine]-9,9′-dicarboxamide    (II-25).

Methods of Making

Examples of synthetic pathways useful for making compounds describedherein are set forth in the Examples below and in Schemes I-VII.

Compound Ic-7 can be synthesized by reacting commercially available3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diamine (1 equivalent) with 1.2equivalents of the commercially available methyl ester of iodobenzoicacid in the presence of Pd₂(dba)₃/DPPP (0.1-0.5 equiv) and 1.2equivalents Cs₂CO₃ in toluene at 110° C. for 20 h with vigorousstirring. The intermediate compound methyl2-((4′-amino-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)benzoate isisolated by flash chromatography on a C18 column. The product is thenreacted by addition of 1.2 equivalent of methyl 2-iodo-5-methylbenzoatein the presence of catalytic (0.1-0.5 equiv) Pd₂(dba)₃/DPPP and 1.2equivalents anhydrous, finely powdered Cs₂CO₃ in toluene at 110° C. for20 h to yield methyl2-((3,3′-dimethoxy-4′-((2-(methoxycarbonyl)phenyl)amino)-[1,1′-biphenyl]-4-yl)amino)-5-methylbenzoate.The methyl esters can then be hydrolyzed under basic conditions followedby acidification to yield compoundIc-7,2-((4′-((2-carboxyphenyl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)-5-methylbenzoicacid.

The intermediate compound from Scheme I, methyl2-((4′-amino-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)benzoate isreacted by addition of 1.2 equivalent of methyl 3-iodoisonicotinate inthe presence of catalytic (0.1 equiv) Pd₂(dba)₃/DPPP and 1.2 equivalentsanhydrous, finely powdered Cs₂CO₃ in toluene at 110° C. for 20 h withvigorous stirring. The methyl esters can be subsequently hydrolyzed byheating the product under basic conditions followed by acidification toyield compoundIc-8,3-((4′-((2-carboxyphenyl)amino)-3,3′-dimethoxy-[1,1′-biphenyl]-4-yl)amino)isonicotinicacid.

The compound Ic-17 can be synthesized by reacting commercially available3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diamine (1 equivalent) with 2.4equivalents of the commercially available methyl 2-iodo-5-methylbenzoatein the presence of Pd₂(dba)₃/DPPP (0.1-0.5 equiv) and 2.4 equivalentsanhydrous, finely powdered Cs₂CO₃ in toluene at 110° C. for 20 h withvigorous stirring. The methyl esters can be subsequently hydrolyzed byheating the product under basic conditions followed by acidification toyield compound Ic-17.

The compound Ic-18 can be synthesized by reacting commercially available3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diamine (1 equivalent) with 2.4equivalents of the commercially available methyl2-iodo-4-methoxybenzoate in the presence of Pd₂(dba)₃/DPPP (0.1-0.5equiv) and 2.4 equivalents anhydrous, finely powdered Cs₂CO₃ in tolueneat 110° C. for 20 h with vigorous stirring. The methyl esters can besubsequently hydrolyzed by heating the product under basic conditionsfollowed by acidification to yield compound Ic-18.

Compound Ic-6 can be prepared by treating 0.2 mmol of the commerciallyavailable starting material2,2′-((3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diyl)bis(azanediyl))dibenzoicacid in a stirred solution of anhydrous CH₂Cl₂ (10 mL) at −78° C. with a20-fold excess of BBr₃ (4 mmol), added dropwise, for 1 h. The solutionis then warmed to room temperature for 1 h. The reaction is quenched byslowly adding 10 mL of an ice cold solution of 6 M HCl to the mixtureand stirring at room temperature for 1 h. The aqueous layer is extractedwith CH₂Cl₂ (3×50 mL), and the combined organic layers are dried overMgSO₄. The solvent is concentrated in vacuo and the residue is purifiedon a C18 column eluted with a linear gradient from 5% acetonitrile. 0.1%TFA to 100% acetonitrile/0.1% TFA.

The compound Ic-1 can be synthesized by reacting commercially available3,3′-dimethoxy-[1,1′-biphenyl]-4,4′-diamine (1 equivalent) with 2.4equivalents of the commercially available methyl 2-iodo-5-methylbenzoatein the presence of Pd₂(dba)₃/DPPP (0.1-0.5 equiv) and 2.4 equivalentsanhydrous, finely powdered Cs₂CO₃ in toluene at 110° C. for 20 h withvigorous stirring. The methyl esters can be subsequently hydrolyzed byheating the product under basic conditions followed by acidification toyield compound Ic-1.

The compound II-19 (prepared as described in Example 1) is converted tothe alcohol form II-20 by dissolving the starting material in DMSO andtreating the compound with 10 equivalents of NaCNBH₃ for 24 h at roomtemperature. The product is purified by reverse phase chromatography ona C18 column eluted with a linear gradient from 5% acetonitrile. 0.1%TFA to 100% acetonitrile/0.1% TFA.

Methods of Using

The invention relates to treating hematological malignancies withpharmaceutical compositions comprising one or more compounds of FormulaIa′, Ib′, Ic′, or II′, and/or BH3 mimic compounds that inhibit a broadrange of the Bcl-2 family of proteins, most notably Mcl-1. It iscontemplated that the activity against the protein Mcl-1 of thecompounds A or II-19 (FIGS. 1-7) as well as derivative compounds willenable therapeutic utility of these compounds as anti-tumor agents intreating cancer, including blood cancers.

The invention, for example, provides a method for treating particulartypes of hematopoietic cancers, using pharmaceutical compositionscomprising BH3 mimic compounds of Formula Ia′, Ib′, Ic′, or II′. The useof these compounds for particular types of hematopoietic cancers mayhave unexpected results in terms of efficacy and/or ability to inhibitparticular anti-apoptotic (pro-survival) members of the Bcl-2 family orto mimic particular members of the pro-apoptotic Bcl-2 family proteins.Accordingly, hematological tumor cells that are hyper-dependent on aparticular member of the Bcl-2 family of proteins will be most affectedby that BH3 mimic which targets that protein.

Compound A is also known as redoxal, which has the following structure:

Compound A may be particularly useful in a method of treatinghematopoietic cancers, by preferentially inhibiting the binding of theactivator BH3 only proteins of the Bcl-2 family to protein Mcl-1. Thislevel of activity (<500 nM) is among the most potent of all reported BH3mimics and directs the use of this compound in treating certainhematological malignancies that are affected principally by the Bcl-2family proteins and among those proteins, mostly by Mcl-1. Based on theunique ability of compound A to inhibit BH3 binding to Mcl-1, thiscompound may be particularly effective in blocking the unwanted cellsurvival activity of Mcl-1 in tumorogenic lymphoid and myeloid cells.This feature of compound A will direct its use as a potentialtherapeutic agent for treating Multiple Myeloma (MM), diffuse largeB-cell lymphoma (DLBCL), chronic lymphocytic leukemia (CLL), acutemyelogenous leukemia (AML), all of which are effected by elevated Mcl-1.Compound II-19 exhibits surprisingly high affinity against Mcl-1 (IC₅₀=7nM) and Bcl-xL (IC₅₀=15 nM) and this level of activity makes it the mostpotent of small molecule (non-peptide) BH3 mimetics reported to date.This potent activity directs its use in treating certain hematologicalmalignancies that are affected by Bcl-2 family proteins that includeMcl-1, Bcl-xL, or the closely protein related Bcl-2. Similarly, activityin derivatives of compound A, compounds of Formula Ia, Ib, Ic, as wellas compounds of Formula II, may direct the use of those compounds intreating lymphoid and myeloid malignancies.

Methods of treatment of hematopoietic cancers in patients usingpharmaceutical compositions comprising compounds of Formula Ia′, Ib′,Ic′, or II′ are provided. These compounds have broad activity againstall Bcl-2 family proteins, most notably including Bcl-2 protein familymember Mcl-1, which is not expected. Accordingly, one aspect of thisinvention is directed to a method for treating hematopoietic cancers,such as Multiple Myeloma, by preferentially targeting Bcl-2 proteinfamily member Mcl-1 with a member of the group consisting ofpharmaceutical compositions comprising compounds of Formula Ia′, Ib′,Ic′, or II′.

Methods for tumor regression and enhancing survival in B-cell lymphomasin patients by administering pharmaceutical compositions comprising acompound of Formula Ia′, Ib′, Ic′, or II′ are provided. Accordingly, inone embodiment, this invention describes a method for using compounds ofFormula Ia, Ib, and Ic for the treatment of non-Hodgkin's B-celllymphoma, including CLL, Burkett's, Indolent and Aggressivenon-Hodgkin's lymphomas, Multiple Myelomas, or other cancers that areaffected by Bcl-2 family of proteins, and in particular the proteinMcl-1.

These treatments may be accomplished utilizing pharmaceuticalcompositions comprising a compound of Formula Ia′, Ib′, Ic′, or II′alone or in combination with other chemotherapy agents or with radiationtherapy. Accordingly, the invention provides a method for treatingparticular types of hematopoietic cancers using a combination of one ormore compounds selected from the group consisting of pharmaceuticalcompositions comprising a compound of Formula Ia′, Ib′, Ic′, or II′, incombination with other therapies, for example, a class of therapeuticsknown as 26S proteosome inhibitors, such as, for example, Bortezomib(Velcade®).

In addition, this invention relates to methods for determiningselectivity of compounds of Formula Ia′, Ib′, Ic′, or II′ and BH3 mimiccompounds to predict efficacy in treating hematological and othermalignancies involving Bcl-2 family proteins. For example, thesecompounds can have varying potencies in inhibiting BH3 mediated-bindingof particular Bcl-2 family proteins, and the difference in potency canbe identified by systematically ordering combinations of protein-proteininteractions and comparing the blocking activity of BH3 mimic compoundsto that of competing BH3 domain-containing peptides. By matching theactivity of the compound to a particular BH3 domain peptide, abiological activity can be assigned to that compound that correlates tothe activity of the BH3 domain-containing protein. This information canbe used to predict the utility of a BH3 mimic compounds in treating aparticular disease.

This invention also relates to cancer treatments and especially toMultiple Myeloma treatments directed to Bcl-2 and Bcl-xL and Mcl-1 andBcl-2A1 and Bcl-w (referred as a group as an anti-apoptotic Bcl-2family) activity.

A. Treatment with Compounds of Formula Ia′, Ib′, Ic′, or II′ to InhibitBcl-2 Proteins

The present invention describes anti-tumor efficacy and enhancedsurvivability in mouse models for hematological malignancies bytreatment with pharmaceutical compositions comprising one or morecompounds of Formula Ia′, Ib′, Ic′, or II′. Efficacy in certain animalmodels may be transferable to humans afflicted with B-cell lymphoma orother hematological or non-hematological cancers affected by Bcl-2family proteins.

This treatment could be administered as a stand-alone therapy or withprior art chemotherapy agents or with radiation therapy. In oneembodiment, the pharmaceutical compositions comprising compounds ofFormula Ia′, Ib′, Ic′, or II′, are used for the treatment of B-celllymphoma or Multiple Myeloma by inducing cancer cell death andpreventing cancer cell migration to spleen or lymph nodes.

Because Mcl-1 has emerged as a key member of the Bcl-2 family ofproteins for initiating and maintaining certain Myeloid as well asB-cell and T-cell malignancies, it is an important target for treatmentof many hematological diseases. This invention demonstrates theeffectiveness of the compound A in inhibiting BH3 binding to Mcl-1. Itis taught that this feature of compound A is useful for treatingMultiple Myeloma, B-cell lymphoma or other hematological cancers orother disease that are affected by Mcl-1 activity including prostate,liver, and ovarian cancers.

Compounds of Formula Ia′, Ib′, Ic′, or II′ have activity against Mcl-1.Accordingly, compounds of Formula Ia′, Ib′, Ic′, or II′ andpharmaceutical compositions comprising a compound of Formula Ia′, Ib′,Ic′, or II′ are useful therapeutic compounds in treating MM, NHL, CLL,AML, prostate, liver and ovarian cancers, and melanomas.

This activity will also direct the use of these compounds for treatmentof certain autoimmune diseases that are affected by excess B or T cellproliferation.

Data described in the examples below indicate that compound A has uniquecapability to preferentially disrupt binding of Bim BH3 peptides topurified Mcl-1 in comparison to Bcl-xL protein in an in vitro assay. Anexperiment reported below used Fluorescence polarization (FP) to assessthe concentration of compound A or compound II-19 that causes 50%inhibition (IC₅₀) of Bim BH3 binding to Mcl-1 (Example 1). Binding ofpeptides consisting of the BH3 binding domain of the pro-apoptoticprotein Bak to purified Human Bcl-xL or Human Mcl-1 was carried out inthe presence of titrated compound A, or compound II-19. (Degterev et al.(2002) Nat. Cell. Biol. 3: 173-82). These compounds were effective atblocking Bim binding to Bcl-xL. However, each is more effective atbinding of Mcl-1 and demonstrated lower IC₅₀. This preferred sensitivityof Mcl-1 for compounds A, or compound II-19, indicates that thesecompounds have novel characteristics that make them uniquely valuable asanti-tumor therapeutics. This activity of compounds of Formula Ia′, Ib′,Ic′, or II′ in inhibiting BH3 mediated binding to Mcl-1 has not beenreported in the literature.

The present invention relates to the use of pharmaceutical compositionscomprising a compound of Formula Ia′, Ib′, Ic′, or II′ as compositionsin inhibiting the activity of Bcl-2 pro-survival proteins, mostparticularly Mcl-1, in tumor cells and thereby killing those cells. Theability of these compounds to inhibit Mcl-1 function in cells makesthese compounds effective anti-B-cell, T-cell, and Myeloma cell cancertherapeutics for treating non-Hodgkin's lymphoma, CLL, MM, and AML aswell as prostate, colon, ovarian, and liver cancer and melanoma.

Compounds of Formula Ia′, Ib′, Ic′, or II′, cause tumor regression, forexample by killing a cancer cell, and increased survival in severalmouse tumor models, including, for example models for diffuse largeB-cell lymphoma (DLBCL) (Cattoretti et al. (2005) Cancer Cell 7:445-55), small B cell lymphoma/CLL (Zapata et al. (2004) Proc. Natl.Acad. Sci. USA 101(47): 16600-5) and migrating B-cell lymphomas (Refaeliet al. (2005) Proc. Natl. Acad. Sci. USA 102(11): 4097-102), as well asan AML mouse tumor model (Lopes de Menezes et al. (2005) Clin. Canc.Res. 11(14): 5281-91). All of the tumors from these cell models arecharacterized as having elevated pro-survival Bcl-2 family proteins,including Bcl-2, Bcl-xL, and Mcl-1.

Accordingly, the present invention relates to the use of pharmaceuticalcompositions comprising a compound of Formula Ia′, Ib′, Ic′, or II′ inaffecting tumor regression in human lymphoid and myeloid cancers. Thesecompounds are effective in inducing apoptosis selectively inhematological cancers due to the hyper-dependence of lymphoid andmyeloid-derived tumor cells on the activity of the Bcl-2 familyanti-apoptotic proteins.

The Bcl-2 protein is a member of an entire family, the Bcl-2 family ofproteins, that have structurally similar genes and that share sequencehomology and participate in the control of programmed cell death or“apoptosis” (Corey et al. (2002) Nat. Rev. Cancer 2: 647-656). Somemembers of this family (anti-apoptotic Bcl-2 family proteins), such asBcl-2 Bcl-xL, BcL-w, Bfl-1(A1) and Mcl-1, protect cells from apoptosis.These proteins share sequence homology in 4-helical regions called theBcl-2 homology (BH)-domains 1-4 (BH1-BH4). Another class of this family(pro-apoptotic Bcl-2 proteins), such as Bax and Bak, promote apoptosisand share three of these domains, BH1-BH3. A third class of Bcl-2 familyproteins, such as Bim, Bad, Hrk, Bid, Puma, Noxa, and Bmf, share onlyone region, the BH3 domain, and are referred to as “BH3-only proteins”.The BH3-only proteins are pro-apoptotic, and like Bax and Bak, theBH3-only proteins require an intact BH3 domain to promote apoptosis(Adams et al. (1998) Science 281: 1322-26).

A complex interplay of the pro-apoptotic and anti-apoptotic proteinsaffects the integrity of the outer membrane of the mitochondria (Greenet al. (2004) Science 305: 626-29) either causing or preventing therelease of certain molecules that activate the cysteine aspartylproteases (caspases). The caspases are the eventual effectors ofapoptosis (Salvesen (2002) Cell Death and Differentiation 9: 3-5). Baxand Bak are essential for release of these apoptosis promoting moleculesfrom the mitochondria (Wei et al. (2001) Science 292: 727-30). TheBH3-only proteins stimulate the activity of Bax and Bak while theanti-apoptotic proteins oppose their activity. Essentially all of theseinteractions occur by BH3 domain mediated binding (Chrittenden et al.(1995) EMBO J. 14:5589-96).

Anti-apoptotic family members Bcl-2, Bcl-xL and Mcl-1 are over-expressedin many types of cancers, including lymphomas, melanomas, myelomas, andcancer in prostate and colon (Kitada et al. (2002) Oncogene 21: 3459-74;Paul-Samojedny et al. (2005) Biochem. Biophys. Acta. 1741(1-2): 25-9;Pollack et al. (2003) Cancer 97(7): 1630-8; Tas et al. (2004) MelanomaRes. 14(6): 543-6). Animal model studies established that the continuouspresence of anti-apoptotic family members is required for tumor survivaland growth. Additionally, the pro-survival Bcl-2 proteins are importantfor the development of resistance of tumor cells to chemotherapies suchas DNA damaging agents. The ratio of pro-apoptotic to anti-apoptoticfamily members has been shown in many cases to hold significantprognostic value for patient outcome. Over-expression of anti-apoptoticBcl-2 family proteins has been reported in many of the hematopoieticmalignancies. For example, increased expression of Bcl-2 protein thatresults from a translocation (t14; 18) of the BCL2 gene occurs in 80% to90% of low-grade follicular non-Hodgkin lymphomas (NHLs) (Kitada et al.(2002) supra).

Three different strategies for countering the tumorigenic effects ofanti-apoptotic Bcl-2 family proteins in NHL, CLL, MM, and other types ofcancer include: (1) inhibiting gene transcription; (2) using antisenseoligonucleotides to cause mRNA degradation; and (3) directly inhibitingthe proteins with small-molecule drugs (reviewed in Reed et al. (2005)Blood 106: 408-418).

One of the desired characteristics of anti-tumor drugs is the ability toinduce apoptosis in tumor cells and not in healthy cells. Theconventional chemotherapy is mostly based on the evidence thatproliferating cells are more sensitive to anticancer agents thannon-dividing cells (Marchini et al. (2004) Curr. Med. Chem. AnticancerAgents 4(3): 247-6). For instance, generally tumor cells are moresensitive to apoptosis induction by microtubule poisons such as taxoland DNA damaging drugs such as doxorubicin than healthy cells (Abal etal. (2003) Curr. Cancer Drug Targets 3(3): 193-203).

However, in many types of cancer, certain of the anti-apoptotic Bcl-2family proteins are elevated which causes cells to be less responsive tosuch drugs. This is especially true in B-cell lymphomas and otherhematological malignancies. In these cancers elevated levels ofanti-apoptotic Bcl-2 family proteins correlate highly with the onset,maintenance of the disease state, and chemoresistance (Kitada et al.(2002) supra).

However, it was reported that cells over-expressing Bcl-xL exhibitedincreased sensitivity to an antimycin-A derivative compound that bindsto and inhibits Bcl-2 and Bcl-xL (Manion et al. (2004) J. Biol. Chem.279(3): 2159-65; Kim et al. (2001) Biochemistry 40: 4911-22). Thisfinding has implications for the use of certain BH3 mimics as anti-tumortherapeutic compounds given that over-expression of Bcl-xL or Bcl-2results in a general decrease in responsiveness to apoptotic cues andhas been implicated in multi-drug resistance in cancer cells andcarcinogenesis.

An understanding of the mechanisms for this observed change in responseto Bcl-2 or Bcl-xL targeted compounds has been described (Letai (2005)J. Clin. Invest. 115: 2648-55). In that report it was argued that thecell context in which elevated anti-apoptotic Bcl-2 proteins are founddetermines the occurrence or the degree of “sensitization” to apoptoticcues. Most notably, it is the presence of BH3-only proteins bound tothese anti-apoptotic proteins that cause sensitization to apoptoticcues. For instance, the presence of the BH3-only protein Bad (Bcl-2associated death promoter) bound to Bcl-2 or Bcl-xL sensitizes ratherthan kills cells, as is the case when the BH3-only protein Bim(Bcl-2-like 11) binds (Letai et al. (2002) Cancer Cell 2(3): 183-92).Other arguments have been put forth that describe “hyper-dependence” oncertain elevated anti-apoptotic Bcl-2 proteins in certain tumor cells(Kim et al. (2001) Biochemistry 40: 4911-22).

The functions that the individual Bcl-2 family proteins have duringhematopoiesis have been demarcated genetically using transgenic mice.For example, mice deficient in Bcl-2 have no overt problems duringlymphocyte differentiation but do have excess apoptosis in peripherallymphocytes after antigenic stimuli (Veis et al. (1993) Cell 75: 229).Bcl-xL deficient mice are also viable but do show late maturation oferythroid cells (Wagner et al. (2000) Development 127: 4949-58).

Mcl-1 deficiency has a pronounced, perhaps principal role, in lymphocytesurvival. Conditional knockouts have been used to determine the role ofMcl-1 in hematopoiesis and lymphocyte survival. It was determined thatconditional deficiency of Mcl-1 results in apoptosis of differentiatinglymphocytes and stops development of pre-B-cell and double negativeT-cells as well as apoptosis in mature B and T lymphocytes (Rinkenbergeret al. (2000) Genes Dev. 14: 23). These findings demonstrate a role forthe anti-apoptotic form of Mcl-1 in the development and survival of Band T lymphocytes. These findings also indicate that Mcl-1 may be anideal target for treating excess growth of lymphoid cells.

The clinical implication is underscored by the observation that elevatedMcl-1 expressed in its active anti-apoptotic full length form positivelycorrelates with increasing grade of B-cell lymphomas and plasma cellmyelomas (Cho-Vega et al. (2004) Hum. Pathol. 35 (9): 1095-100) as wellas chronic lymphocytic leukemia (Petlickovski et al. (2005) Blood 105:4820-28).

Targeted gene knockouts for different pro-apoptotic BH3-only members ofthe Bcl-2 family members have been assessed for disease correlation.Transgenic mice deficient in Bim have extensive myeloid proliferationand autoreactive T and B cells that have lost responsiveness toapoptosis inducing drugs (Bouillet et al. (1999) Science 286: 1735-38),while mice deficient in Bad display high incidence of diffuse large celllymphoma (Ranger et al. (2003) Proc. Natl. Acad. Sci. USA 100: 9324-29).Mice deficient in the pro-apoptotic BH3-only protein Bid demonstratedHepatocarcinoma and a failure to respond to the death inducing cytokinefas (Yin et al. (1999) Nature 400: 886-891). Both of the pro-apoptoticBH3-only proteins Puma and Noxa were shown to be required for allp53-mediated apoptosis (Villunger et al. (2003) Science 302: 1036-1040).

Notably, conditional knockouts of the Mcl-1 gene caused profoundreduction in B and T lymphocytes (Opferman et al. (2003) Nature426(6967): 671-6), which is the opposite of a deficiency in the BH3-onlyprotein Bim and in keeping with the understanding that Mcl-1 selectivelyinhibits the pro-apoptotic protein Bim.

B. Combination Therapy

Embodiments of the present invention also include the combination of oneor more compounds selected from the group consisting of compounds ofFormula Ia′, Ib′, Ic′, or II′ with other anti-tumor agents, such asproteosome inhibitors, to yield combination therapies. In someinstances, these combination therapies may yield synergistic results ascompared to the additive results of the component therapies when usedalone. For example, these compounds may be particularly effective whenused in combination with a class of therapeutics known as 26S proteosomeinhibitors.

Compounds that have activity as 26S proteosome inhibitors have beensuggested for use as anti-tumor therapeutics based on their ability toinhibit Nf-Kb signaling (Li et al. (1995) Biophys. Biochem. Res. Com.215: 292-301). One such compound, the FDA approved drug Bortezomib(Velcade®), has been shown to cause elevated Mcl-1 in lymphocytes(Nencioni et al. (2005) Blood 105(8): 3255-62). Elevated Mcl-1 has beenshown to be causal in the establishment and maintenance of lymphoid andmyeloid tumors. The unwanted side effect of elevated Mcl-1 can berectified by inhibiting Mcl-1 using the compounds of Formula Ia′, Ib′,Ic′, or II′ will have utility in potentiating the effect of Bortezomibor other 26S proteosome inhibitors as anti-tumor therapeutics.

These observations suggest that elevated Mcl-1 may counteract Bortezomib(Velcade®) in CLL, AML, and certain NHL cells. Consistent with this, thecytotoxic effects of proteosome inhibitors are enhanced when Mcl-1levels are contained at normal levels or reduced in a cell culture(Nencioni A et al. (2005) supra). This finding demonstrated that Mcl-1accumulation is an unwanted molecular consequence of exposure toproteosome inhibitors.

C. Screening Methods

The present invention teaches a method for selecting appropriate BH3mimic compounds in treating particular tumors. This selection is basedon an understanding of the unique activity of compounds of Formula Ia′,Ib′, Ic′, or II′ in mimicking particular BH3 domains. It is taught thatcompounds from this group that have unique activity against either allof the anti-apoptotic Bcl-2 family proteins or a particular member ofthis family of proteins will be the basis for use against particulartumors. Expression levels of particular Bcl-2 family proteins can beassessed using standard assays, western blot, or immunohistologicalstaining of biopsied tumor tissue. Following this assessment, compoundswith activity against the elevated proteins in the tumor sample will beselected as appropriate therapeutics for treating that tumor.

It is of particular interest to establish the correlation of Mcl-1expression levels to the occurrence of tumors. Based on the discoverythat compounds A or II-19 inhibit Mcl-1 binding (see Example 2 andExample 3 below), it may be that cells that are hyper-dependent onMcl-1, as a consequence of elevated Mcl-1, or elevated levels of Mcl-1complexed with Bim, Bid or Noxa, in the disease state, will besensitized to compounds of Formula Ia′, Ib′, Ic′, or II′ or other BH3mimic compounds that are shown to inhibit Mcl-1.

D. Administration and Dosage

i. Routes of Administration

The pharmaceutical compositions comprising a compound of Formula Ia′,Ib′, Ic′, or II′ can be administered by any known administration methodknown to a person skilled in the art. Examples of routes ofadministration include but are not limited to oral, parenteral,intraperitoneal, intravenous, intraarterial, transdermal, topical,sublingual, intramuscular, rectal, transbuccal, intranasal, liposomal,via inhalation, vaginal, intraoccular, via local delivery by catheter orstent, subcutaneous, intraadiposal, intraarticular, intrathecal, or in acontrolled or extended release dosage form. The pharmaceuticalcompositions comprising a compound of Formula Ia′, Ib′, Ic′, or II′ canbe administered in accordance with any dose and dosing schedule thatachieves a dose effective to treat disease.

The route of administration of pharmaceutical compositions comprising acompound of Formula Ia′, Ib′, Ic′, or II′ can be independent of theroute of administration of any additional anti-cancer agents that areused. Thus, at least one of the compounds of Formula Ia′, Ib′, Ic′, orII′ can be administered, for example, orally or by intravenous deliverywhile another compound or other agent (anti-cancer agent) can beadministered, for example, orally, parenterally, intraperitoneally,intravenously, intraarterially, transdermally, sublingually,intramuscularly, rectally, transbuccally, intranasally, liposomally, viainhalation, vaginally, intraoccularly, via local delivery by catheter orstent, subcutaneously, intraadiposally, intraarticularly, intrathecally,or in a controlled or extended release dosage form.

As examples, the compounds of the invention can be administered in oralforms, for example, as tablets, capsules (each of which includessustained release or timed release formulations), pills, powders,granules, elixirs, tinctures, suspensions, syrups, and emulsions.Likewise, the compounds can be administered by intravenous (e.g., bolusor infusion), intraperitoneal, subcutaneous, intramuscular, or otherroutes using forms well known to those of ordinary skill in thepharmaceutical arts. Particularly useful routes of administration of thecompounds are oral administration and intravenous delivery.

The compounds can also be administered in the form of a depot injectionor implant preparation, which may be formulated in such a manner as topermit a sustained release of the active ingredient. The activeingredient can be compressed into pellets or small cylinders andimplanted subcutaneously or intramuscularly as depot injections orimplants. Implants may employ inert materials such as biodegradablepolymers or synthetic silicones, for example, Silastic, silicone rubberor other polymers manufactured by the Dow-Corning Corporation.

The compounds can also be delivered by the use of monoclonal antibodiesas individual carriers to which the compound molecules are coupled.

The compounds can also be prepared with soluble polymers as targetabledrug carriers. Such polymers can include polyvinlypyrrolidone, pyrancopolymer, polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds can beprepared with biodegradable polymers useful in achieving controlledrelease of a drug, for example, polylactic acid, polyglycolic acid,copolymers of polylactic and polyglycolic acid, polyepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross linked or amphipathicblock copolymers of hydrogels.

In a specific embodiment, the compounds can be administered orally in agelatin capsule, which can comprise excipients such as microcrystallinecellulose, croscarmellose sodium and magnesium stearate. For example, anembodiment can include 200 mg of solid compound with 89.5 mg ofmicrocrystalline cellulose, 9 mg of sodium croscarmellose, and 1.5 mg ofmagnesium stearate contained in a gelatin capsule.

ii. Dosages and Dosage Schedules

The dosage regimen utilizing the compounds of Formula Ia′, Ib′, Ic′, orII′ can be selected in accordance with a variety of factors includingtype, species, age, weight, and sex of the patient; the type of diseasebeing treated; the severity (i.e., stage) of the disease to be treated;the route of administration; the renal and hepatic function of thepatient; and the particular compound or salt thereof employed. A dosageregimen can be used, for example, to prevent, inhibit (fully orpartially), or arrest the progress of the disease.

In accordance with the invention, pharmaceutical compositions comprisinga compound of Formula Ia′, Ib′, Ic′, or II′ can be administered bycontinuous or intermittent dosages. For example, intermittentadministration of pharmaceutical compositions comprising a compound ofFormula Ia′, Ib′, Ic′, or II′ may be administered one to six days perweek or it may be administered in cycles with rest periods in betweenthe cycles (e.g., daily administration for two to eight consecutiveweeks, then a rest period with no administration for up to one weekbetween treatments) or it may be administered on alternate days.

For example, the compounds of Formula Ia′, Ib′, Ic′, or II′ can beadministered in a total daily dose of up to 800 mg. The compounds ofFormula Ia′, Ib′, Ic′, or II′ can be administered once daily (QD), ordivided into multiple daily doses such as twice daily (BID), and threetimes daily (TID). The compounds of Formula Ia′, Ib′, Ic′, or II′, canbe administered at a total daily dosage of up to 800 mg, for example,about 200 mg, 300 mg, 400 mg, 600 mg, or 800 mg, which can beadministered in one daily dose or can be divided into multiple dailydoses as described above. In specific aspects, the administration isoral or by intravenous delivery.

In one embodiment, the compound is administered once daily at a dose ofabout 200-600 mg. In another embodiment, the compound is administeredtwice daily at a dose of about 200-400 mg. In another embodiment, thecompound is administered twice daily at a dose of about 200-400 mgintermittently, for example three, four or five days per week. In oneembodiment, the daily dose is about 200 mg which can be administeredonce-daily, twice-daily, or three-times daily. In one embodiment, thedaily dose is about 300 mg which can be administered once-daily,twice-daily, or three-times daily. In one embodiment, the daily dose isabout 400 mg which can be administered once-daily, twice-daily, orthree-times daily.

Compounds of Formula Ia′, Ib′, Ic′, or II′, can be administered inaccordance with any dose and dosing schedule that achieves a doseeffective to treat cancer. Each compound can be administered in a totaldaily dose that may vary from patient to patient, and may beadministered at varying dosage schedules. For example, a compound of theinvention can be administered to the patient at a total daily dosage ofbetween 25-4000 mg/m². In particular, compounds of Formula Ia′, Ib′,Ic′, or II′ can be administered in a total daily dose of up to 800 mg,especially by oral or intravenous administration, once, twice, or threetimes daily, continuously (every day) or intermittently (e.g., 3-5 daysa week).

In addition, the compounds of Formula Ia′, Ib′, Ic′, or II′ may beadministered according to any of the schedules described above,consecutively for a few weeks, followed by a rest period.

In one embodiment, the patient can receive intravenously orsubcutaneously the compounds of Formula Ia′, Ib′, Ic′, or II′ inquantities sufficient to deliver between about 3-1500 mg/m² per day, forexample, about 3, 30, 60, 90, 180, 300, 600, 900, 1200 or 1500 mg/m² perday. Such quantities may be administered in a number of suitable ways,e.g. large volumes of low concentrations of the compounds or FormulaIa′, Ib′, Ic′, or II′ can be used during one extended period of time orseveral times a day. The quantities can be administered for one or moreconsecutive days, intermittent days, or a combination thereof per week(7 day period). Alternatively, low volumes of high concentrations of thecompounds of Formula Ia′, Ib′, Ic′, or II′ can be used during a shortperiod of time, e.g. once a day for one or more days eitherconsecutively, intermittently or a combination thereof per week (7 dayperiod). For example, a dose of 300 mg/m² per day can be administeredfor 5 consecutive days for a total of about 1500 mg/m² per treatment. Inanother dosing regimen, the number of consecutive days can also be 5,with treatment lasting for 2 or 3 consecutive weeks for a total of about3000 mg/m² or about 4500 mg/m² total treatment.

Typically, an intravenous formulation may be prepared which contains aconcentration of compounds of Formula Ia′, Ib′, Ic′, or II′ of betweenabout 1.0 mg/mL to about 10 mg/mL, e.g. about 2.0 mg/mL, 3.0 mg/mL, 4.0mg/mL, 5.0 mg/mL, 6.0 mg/mL, 7.0 mg/mL, 8.0 mg/mL, 9.0 mg/mL and 10mg/mL and administered in amounts to achieve the doses described above.In one example, a sufficient volume of intravenous formulation can beadministered to a patient in a day such that the total dose for the dayis between about 300 and about 1500 mg/m².

Subcutaneous formulations can be prepared according to procedures wellknown in the art at a pH in the range between about 5 and about 12,which include suitable buffers and are tonicity agents, as describedbelow. They can be formulated to deliver a daily dose of any ofcompounds of Formula Ia′, Ib′, Ic′, or II′ in one or more dailysubcutaneous administrations, for example, one, two or three times eachday.

It is apparent to a person skilled in the art that any one or more ofthe specific dosages and dosage schedules of the compounds of FormulaIa′, Ib′, Ic′, or II′ are also applicable to any one or more of theanti-cancer agents to be used in a combination treatment. Moreover, thespecific dosage and dosage schedule of the compounds of Formula Ia′,Ib′, Ic′, or II′ can further vary, and the optimal dose, dosingschedule, and route of administration can be determined based upon thespecific drug combination that is being used. Further, the various modesof administration, dosages, and dosing schedules described herein merelyset forth specific embodiments and should not be construed as limitingthe broad scope of the invention. Any permutations, variations, andcombinations of the dosages and dosing schedules are included within thescope of the present invention.

iii. Formulation

An “effective amount” of compounds of Formula Ia′, Ib′, Ic′, or II′ isthe quantity which, when administered to a subject having a disease ordisorder, results in regression of the disease or disorder in thesubject. Thus, an effective amount of a compound of the disclosedinvention is the quantity which, when administered to a subject having acell proliferation disorder, results in, for example, regression of cellgrowth or cell death in a subject. The amount of the disclosed compoundto be administered to a subject will depend on the particular disorder,the mode of administration, co-administered compounds, if any, and thecharacteristics of the subject, such as general health, other diseases,age, sex, genotype, body weight and tolerance to drugs. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors.

As used herein, the term “effective amount” refers to an amount of acompound, or a combination of compounds, of the present inventioneffective when administered alone or in combination as ananti-proliferative agent. For example, an effective amount refers to anamount of the compound present in a formulation or on a medical devicegiven to a recipient patient or subject sufficient to elicit biologicalactivity, for example, anti-proliferative activity, such as for example,anti-cancer activity or anti-neoplastic activity. The combination ofcompounds optionally is a synergistic combination. Synergy, asdescribed, for example, by Chou and Talalay (1984) Adv. Enzyme Regul.22: 27-55, occurs when the effect of the compounds when administered incombination is greater than the additive effect of the compounds whenadministered alone as a single agent. In general, a synergistic effectis most clearly demonstrated at sub-optimal concentrations of thecompounds. Synergy can be in terms of lower cytotoxicity, or increasedanti-proliferative effect, or some other beneficial effect of thecombination compared with the individual components.

A “therapeutically effective amount” means the amount of a compoundthat, when administered to a mammal for treating a disease, issufficient to effect such treatment for the disease. The“therapeutically effective amount” will vary depending on the compound,the disease and its severity and the age, weight, etc., of the mammal tobe treated.

A therapeutically effective amount of one or more of the compounds canbe formulated with a pharmaceutically acceptable carrier foradministration to a human or an animal. Accordingly, the compounds orthe formulations can be administered, for example, via oral, parenteral,or topical routes, to provide an effective amount of the compound. Inalternative embodiments, the compounds prepared in accordance with thepresent invention can be used to coat or impregnate a medical device,e.g., a stent.

The term “prophylactically effective amount” means an effective amountof a compound or compounds, of the present invention that isadministered to prevent or reduce the risk of unwanted cellularproliferation.

“Pharmacological effect” as used herein encompasses effects produced inthe subject that achieve the intended purpose of a therapy. In onepreferred embodiment, a pharmacological effect means that primaryindications of the subject being treated are prevented, alleviated, orreduced. For example, a pharmacological effect would be one that resultsin the prevention, alleviation or reduction of primary indications in atreated subject. In another preferred embodiment, a pharmacologicaleffect means that disorders or symptoms of the primary indications ofthe subject being treated are prevented, alleviated, or reduced. Forexample, a pharmacological effect would be one that results in theprevention or reduction of primary indications in a treated subject.

A “pharmaceutical composition” is a formulation containing the compoundsof Formula Ia′, Ib′, Ic′, or II′ in a form suitable for administrationto a subject. In one embodiment, the pharmaceutical composition is inbulk or in unit dosage form. The unit dosage form is any of a variety offorms, including, for example, a capsule, an IV bag, a tablet, a singlepump on an aerosol inhaler, or a vial. The quantity of active ingredient(e.g., a formulation of the disclosed compound or salt, hydrate,solvate, or isomer thereof) in a unit dose of composition is aneffective amount and is varied according to the particular treatmentinvolved. One skilled in the art will appreciate that it is sometimesnecessary to make routine variations to the dosage depending on the ageand condition of the patient. The dosage will also depend on the routeof administration. A variety of routes are contemplated, including oral,pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous,intramuscular, intraperitoneal, inhalational, buccal, sublingual,intrapleural, intrathecal, intranasal, and the like. Dosage forms forthe topical or transdermal administration of a compound of thisinvention include powders, sprays, ointments, pastes, creams, lotions,gels, solutions, patches and inhalants. In a preferred embodiment, theactive compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

The compounds of Formula Ia′, Ib′, Ic′, or II′ are capable of furtherforming salts. All of these forms are also contemplated within the scopeof the claimed invention.

“Pharmaceutically acceptable salt” of a compound means a salt that ispharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound. As used herein,“pharmaceutically acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines, alkali or organic salts of acidicresidues such as carboxylic acids, and the like. The pharmaceuticallyacceptable salts include the conventional non-toxic salts or thequaternary ammonium salts of the parent compound formed, for example,from non-toxic inorganic or organic acids. For example, suchconventional non-toxic salts include, but are not limited to, thosederived from inorganic and organic acids selected from 2-acetoxybenzoic,2-hydroxyethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic,bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethanesulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic,glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic,hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic,lactic, lactobionic, lauryl sulfonic, maleic, malic,

mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples include hexanoic acid, cyclopentane propionic acid,pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamicacid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid,4-toluenesulfonic acid, camphorsulfonic acid,4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylic acid, 3-phenylpropionicacid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, andthe like. The invention also encompasses salts formed when an acidicproton present in the parent compound either is replaced by a metal ion,e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; orcoordinates with an organic base such as ethanolamine, diethanolamine,triethanolamine, tromethamine, N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The pharmaceutically acceptable salts of the compounds of Formula Ia′,Ib′, Ic′, or II′ can be synthesized from a parent compound that containsa basic or acidic moiety by conventional chemical methods. Generally,such salts can be prepared by reacting the free acid or base forms ofthese compounds with a stoichiometric amount of the appropriate base oracid in water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in Remington's Pharmaceutical Sciences, 18th ed. (Mack PublishingCompany, 1990). For example, salts can include, but are not limited to,the hydrochloride and acetate salts of the aliphatic amine-containing,hydroxyl amine-containing and imine-containing compounds of the presentinvention.

The compounds of Formula Ia′, Ib′, Ic′, or II′ can also be prepared asesters, for example pharmaceutically acceptable esters. For example acarboxylic acid functional group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl, or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., an acetate, propionate, or other ester.

The compounds of Formula Ia′, Ib′, Ic′, or II′ can also be prepared asprodrugs, for example pharmaceutically acceptable prodrugs. The terms“pro-drug” and “prodrug” are used interchangeably herein and refer toany compound that releases an active parent drug in vivo. Since prodrugsare known to enhance numerous desirable qualities of pharmaceuticals(e.g., solubility, bioavailability, manufacturing, etc.) the compoundsof the present invention can be delivered in prodrug form. Thus, thepresent invention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs of thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxyl, amino,sulfhydryl, carboxyl, or carbonyl group is bonded to any group that maybe cleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxyl or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates, andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxyl functional groups, ester groups (e.g. ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g. N-acetyl), N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention including compounds A, or B or derivatives, and the like,(see, Bundegaard, H. “Design of Prodrugs” p1-92, Elesevier, NewYork-Oxford (1985)).

All percentages and ratios used herein, unless otherwise indicated, areby weight.

“Combination therapy” (or “co-therapy”) includes the administration ofcompounds of Formula Ia′, Ib′, Ic′, or II′ and at least a second agentas part of a specific treatment regimen intended to provide thebeneficial effect from the co-action of these therapeutic agents. Thebeneficial effect of the combination includes, but is not limited to,pharmacokinetic or pharmacodynamic co-action resulting from thecombination of therapeutic agents. Administration of these therapeuticagents in combination typically is carried out over a defined timeperiod (usually minutes, hours, days or weeks depending upon thecombination selected). “Combination therapy” may, but generally is not,intended to encompass the administration of two or more of thesetherapeutic agents as part of separate monotherapy regimens thatincidentally and arbitrarily result in the combinations of the presentinvention.

“Combination therapy” is intended to embrace administration of thesetherapeutic agents in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of thetherapeutic agents, in a substantially simultaneous manner.Substantially simultaneous administration can be accomplished, forexample, by administering to the subject a single capsule having a fixedratio of each therapeutic agent or in multiple, single capsules for eachof the therapeutic agents. Sequential or substantially simultaneousadministration of each therapeutic agent can be effected by anyappropriate route including, but not limited to, oral routes,intravenous routes, intramuscular routes, and direct absorption throughmucous membrane tissues. The therapeutic agents can be administered bythe same route or by different routes. For example, a first therapeuticagent of the combination selected may be administered by intravenousinjection while the other therapeutic agents of the combination may beadministered orally. Alternatively, for example, all therapeutic agentsmay be administered orally or all therapeutic agents may be administeredby intravenous injection. The sequence in which the therapeutic agentsare administered is not narrowly critical.

“Combination therapy” also embraces the administration of thetherapeutic agents as described above in further combination with otherbiologically active ingredients and non-drug therapies (e.g., surgery orradiation treatment). Where the combination therapy further comprises anon-drug treatment, the non-drug treatment may be conducted at anysuitable time so long as a beneficial effect from the co-action of thecombination of the therapeutic agents and non-drug treatment isachieved. For example, in appropriate cases, the beneficial effect isstill achieved when the non-drug treatment is temporally removed fromthe administration of the therapeutic agents, perhaps by days or evenweeks.

Throughout the description, where compositions are described as having,including, or comprising specific components, it is contemplated thatcompositions also consist essentially of, or consist of, the recitedcomponents. Similarly, where processes are described as having,including, or comprising specific process steps, the processes alsoconsist essentially of, or consist of, the recited processing steps.Further, it should be understood that the order of steps or order forperforming certain actions are immaterial so long as the inventionremains operable. Moreover, two or more steps or actions may beconducted simultaneously.

The compounds of Formula Ia′, Ib′, Ic′, or II′, or pharmaceuticallyacceptable salts thereof, can be administered orally, nasally,transdermally, pulmonary, inhalationally, buccally, sublingually,intraperintoneally, subcutaneously, intramuscularly, intravenously,rectally, intrapleurally, intrathecally and parenterally. In certainembodiments, the compound is administered orally. One skilled in the artwill recognize the advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

In one embodiment, compounds of Formula Ia′, Ib′, Ic′, or II′ areprepared for oral administration, wherein the disclosed compounds orsalts thereof are combined with a suitable solid or liquid carrier ordiluent to form capsules, tablets, pills, powders, syrups, solutions,suspensions and the like.

The tablets, pills, capsules, and the like contain from about 1 to about99 weight percent of the active ingredient and a binder such as gumtragacanth, acacias, corn starch or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch or alginic acid; a lubricant such as magnesium stearate; and/or asweetening agent such as sucrose, lactose, saccharin, xylitol, and thelike. When a dosage unit form is a capsule, it often contains, inaddition to materials of the above type, a liquid carrier such as fattyoil.

In some embodiments, various other materials are present as coatings orto modify the physical form of the dosage unit. For instance, in someembodiments, tablets are coated with shellac, sugar or both. In someembodiments, a syrup or elixir contains, in addition to the activeingredient, sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and a flavoring such as cherry or orange flavor,and the like.

For some embodiments relating to parental administration, the compoundsof Formula Ia′, Ib′, Ic′, or II′ or salts, solvates, tautomers orpolymorphs thereof, can be combined with sterile aqueous or organicmedia to form injectable solutions or suspensions. Injectablecompositions are preferably aqueous isotonic solutions or suspensions.The compositions may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

For example, injectable solutions are produced using solvents such assesame or peanut oil or aqueous propylene glycol, as well as aqueoussolutions of water-soluble pharmaceutically-acceptable salts of thecompounds. In some embodiments, dispersions are prepared in glycerol,liquid polyethylene glycols and mixtures thereof in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. The terms “parenteraladministration” and “administered parenterally” as used herein meansmodes of administration other than enteral and topical administration,usually by injection, and includes, without limitation, intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal andintrasternal injection and infusion.

For rectal administration, suitable pharmaceutical compositions are, forexample, topical preparations, suppositories or enemas. Suppositoriesare advantageously prepared from fatty emulsions or suspensions. Thecompositions may be sterilized and/or contain adjuvants, such aspreserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

In some embodiments, the compounds of Formula Ia′, Ib′, Ic′, or II′ areformulated to deliver the active agent by pulmonary administration,e.g., administration of an aerosol formulation containing the activeagent from, for example, a manual pump spray, nebulizer or pressurizedmetered-dose inhaler. In some embodiments, suitable formulations of thistype also include other agents, such as antistatic agents, to maintainthe disclosed compounds as effective aerosols.

A drug delivery device for delivering aerosols comprises a suitableaerosol canister with a metering valve containing a pharmaceuticalaerosol formulation as described and an actuator housing adapted to holdthe canister and allow for drug delivery. The canister in the drugdelivery device has a headspace representing greater than about 15% ofthe total volume of the canister. Often, the polymer intended forpulmonary administration is dissolved, suspended or emulsified in amixture of a solvent, surfactant and propellant. The mixture ismaintained under pressure in a canister that has been sealed with ametering valve.

For nasal administration, either a solid or a liquid carrier can beused. The solid carrier includes a coarse powder having particle size inthe range of, for example, from about 20 to about 500 microns and suchformulation is administered by rapid inhalation through the nasalpassages. In some embodiments where the liquid carrier is used, theformulation is administered as a nasal spray or drops and includes oilor aqueous solutions of the active ingredients.

Also contemplated are formulations that are rapidly dispersing dosageforms, also known as “flash dose” forms. In particular, some embodimentsof the present invention are formulated as compositions that releasetheir active ingredients within a short period of time, for example,typically less than about five minutes, preferably less than aboutninety seconds, more preferably in less than about thirty seconds andmost preferably in less than about ten or fifteen seconds. Suchformulations are suitable for administration to a subject via a varietyof routes, for example by insertion into a body cavity or application toa moist body surface or open wound.

Typically, a “flash dosage” is a solid dosage form that is administeredorally, which rapidly disperses in the mouth, and hence does not requiregreat effort in swallowing and allows the compound to be rapidlyingested or absorbed through the oral mucosal membranes. In someembodiments, suitable rapidly dispersing dosage forms are also used inother applications, including the treatment of wounds and other bodilyinsults and diseased states in which release of the medicament byexternally supplied moisture is not possible.

“Flash dose” forms are known in the art; see for example, effervescentdosage forms and quick release coatings of insoluble microparticles inU.S. Pat. Nos. 5,578,322 and 5,607,697; freeze dried foams and liquidsin U.S. Pat. Nos. 4,642,903 and 5,631,023; melt spinning of dosage formsin U.S. Pat. Nos. 4,855,326, 5,380,473 and 5,518,730; solid, free-formfabrication in U.S. Pat. No. 6,471,992; saccharide-based carrier matrixand a liquid binder in U.S. Pat. Nos. 5,587,172, 5,616,344, 6,277,406,and 5,622,719; and other forms known to the art.

The compounds of Formula Ia′, Ib′, Ic′, or II′ can also be alsoformulated as “pulsed release” formulations, in which the compound isreleased from the pharmaceutical compositions in a series of releases(i.e., pulses). The compounds are also formulated as “sustained release”formulations in which the compound is continuously released from thepharmaceutical composition over a prolonged period.

Also contemplated are formulations, for example, liquid formulations,including cyclic or acyclic encapsulating or solvating agents, forexample, cyclodextrins, polyethers, or polysaccharides (e.g.,methylcellulose), or polyanionic-cyclodextrin derivatives with a sodiumsulfonate salt group separate from the lipophilic cavity by an alkylether spacer group or polysaccharides. In one embodiment, the agent canbe polyanionic β-cyclodextrin derivative with a sodium sulfonate saltseparated from the lipophilic cavity by a butyl ether spacer group,e.g., CAPTISOL® (CyDex, Overland, and KS). One skilled in the art canevaluate suitable agent/disclosed compound formulation ratios bypreparing a solution of the agent in water, e.g., a 40% by weightsolution; preparing serial dilutions, e.g. to make solutions of 20%, 10,5%, 2.5%, 0% (control), and the like; adding an excess (compared to theamount that can be solubilized by the agent) of the disclosed compound;mixing under appropriate conditions, e.g., heating, agitation,sonication, and the like; centrifuging or filtering the resultingmixtures to obtain clear solutions; and analyzing the solutions forconcentration of the disclosed compound.

The disclosure is further illustrated by the following examples, whichare not to be construed as limiting this disclosure in scope or spiritto the specific procedures herein described. It is to be understood thatthe examples are provided to illustrate certain embodiments and that nolimitation to the scope of the disclosure is intended thereby. It is tobe further understood that resort may be had to various otherembodiments, modifications, and equivalents thereof which may suggestthemselves to those skilled in the art without departing from the spiritof the present disclosure and/or scope of the appended claims.

EXAMPLES Example 1 Synthesis of Compound II-19

The starting compound (lomofungin) was dissolved in DMSO and irradiatedwith UV light for 24 hours. Dimerization was monitored by LC-MS on a C18column eluted with a methanol gradient (0-90% methanol with 0.1%trifluoroacetic acid).

The starting material (lomofungin) was characterized by NMR and LC/MS.NMR (DMSO-d₆) gave peaks at 3.99 ppm (3H), 6.84 ppm (1H), 7.27 (1H);8.17 (1H), 10.93 (1H), and 11.10 (1H). LC-MS peak exhibited peaks at 315(MH⁺) and 316 (MH₂ ⁺).

Analysis of Compound II-19 Compound II-19 was characterized by NMR andLC/MS. NMR (DMSO-d₆) gave peaks at 3.99 ppm (3H), 7.34 (1H); 8.21 (1H),11.07 (1H), and 11.21 (1H). LC-MS peak exhibited peaks at 627 (MH⁺) and628 (MH²⁺).

Example 2 Preferential Inhibition of Mcl-1 by Compound A

The expression level of Mcl-1 correlates directly to chemo-sensitivityand survival of certain non-Hodgkin's lymphomas (Petlickovski et al.(2005) Blood 105(12): 4820-7) as well as prostate cancer (Royuela et al.(2001) Eur. Cytokine Netw. 12(4): 654-63), liver cancer (Fleischer etal. (2006) Int. J. Oncol. 28(1): 25-32) and other cancers. Mcl-1 istherefore an ideal target for treating these cancers. This example showsthat the BH3 mimic compound A inhibits the binding of the BH3 domain ofthe Bcl-2 family protein Bim to Mcl-1 and, to a lesser extent, Bcl-xL.Accordingly, this example indicates that compound A may be particularlyeffective in treating certain hematological malignancies that areaffected principally by the Bcl-2 family protein Mcl-1.

Materials and Methods

In this example, a Fluorescence Polarization (FP) assay was used todemonstrate the activity of the BH3 mimic compounds A in inhibitingMcl-1 as well as Bcl-xL binding to Bim BH3 as described in Degterev etal. (2001) Nature Cell Biology 3: 173-182.

A nineteen amino acid peptide, corresponding to the BH3 domain of Bim,with the sequence FITC-GGGIAQELRRIGDEFNAY (SEQ ID NO: 1) was labeledwith the fluorophore FITC according to the manufacturer's instructions(Molecular Probes, Eugene, Oreg.). This sequence was identified as beingable to bind to purified Bcl-xL protein (Sattler et al. (1997) Science275(5302): 983-86) and to have biological activity in cells (Holinger etal. J. Biol. Chem. 274: 13298-1330).

In addition, recombinant GST-Mcl-1 and GST-Bcl-xL fusion proteins weregenerated in E. coli and purified using glutathione-sepharose beadsusing conventional techniques known to those skilled in the fields ofbiochemistry and molecular biology. (methods for preparation aredescribed in Strategies for Protein Purification and Characterization,Marshak, D, Kadonga, J, Burgess R, Knuth, M, Brennan, W, Sue-Hwa, L, CSHpress, Cold Spring Harbor, N.Y.). Binding of the recombinant proteins tothe fluorescent Bim BH3 domain was confirmed by titration of increasingconcentrations of the recombinant proteins against a constant amount oflabeled Bim peptide (16.65 nM). Quantitation of binding was accomplishedby FP assay with mP measurements made on the Analyst-GT reader(Molecular Devices, Sunnyvale, Calif.).

Further, the ability of compound A, to disrupt the interaction betweenthe fluorescent Bim BH3 peptide and the two fusion proteins was assessedusing a fluorescence polarization assay. The Bim peptide (in solution at4 nano-Molar) and either GST-Mcl-1 (in solution at 12.5 nM) orGST-Bcl-xL (in solution at 11.8 nM) were first combined together inphosphate buffered saline buffer, and then the compound solution in DMSOwas added to final concentrations ranging from 10 μM to 0.1 nM).Normally the unbound Bim polypeptide results in polarization of 5 mPunits. Upon binding of the GST-Bcl-xL and GST-Mcl-1 fusion protein,polarization increased to 100 mP units.

Results

Data from Fluorescence polarization studies comparing binding of theBim-BH3-FITC peptide to soluble Mcl-1-GST and Bcl-xL-GST proteinsexposed to compound A in concentrations ranging from 10 μM to 1 nM isshown in FIG. 1. Y axis represents % shift in polarization (mp units).Compound A was effective at inhibiting the Bim-BH3 peptide from theBcl-xL with a drug concentration that provokes a response halfwaybetween baseline and maximum (IC₅₀) of 480 nM for Mcl-1. Also, thecompound was able to inhibit the binding of the Bim BH3 peptide from theBcl-xL protein with an IC₅₀ of >16,000 nM (FIG. 1). As seen in FIG. 1,Bim BH3/Mcl-1 inhibition was significantly more pronounced thanBim/Bcl-xL. This indicates a highly preferential activity of thecompound A which has important implications for the therapeutic value ofthis compound.

Example 3 Inhibition of Mcl-1 by Compound II-19

The compound II-19 is assessed for ability to inhibit binding of the BH3peptide of Bim to Mcl-1 and Bcl-xL. The activity for blocking Bim BH3binding to both Bcl-xL and Mcl-1 fusion proteins are compared as inExample 2.

Materials and Methods

Experiments are performed using the fluorescence polarization assay asdescribed in Example 2. Either Bcl-xL or Mcl-1 fusion proteins weretitrated and maximum mp shift indicate binding concentrations for drugtitration studies, as described in Example 2. Compound II-19, wastitrated from 10 μM to 0.1 nM in 2 fold serial dilution into thepeptide/protein solution. The IC₅₀ for blocking each BH3 peptide fromMcl-1 are assessed using FP, as described in Example 2.

Results

Data from Fluorescence polarization studies comparing binding of theBim-BH3-FITC peptide to soluble Mcl-1-GST and Bcl-xL-GST proteinsexposed to compound II-19 in concentrations ranging from 10 μM to 1 nMis shown in FIG. 2. Y axis represents % shift in polarization (mpunits). Compound II-19 with a drug concentration that provokes aresponse halfway between baseline and maximum (IC₅₀) of 7 nM for Mcl-1and 15 nM for Bcl-xL (FIG. 2). Also compound II-19 has a 2-foldpreference for inhibition of Mcl-1 vs. Bcl-xL as seen in FIG. 2. Thecompound II-19 is more active against Mcl-1 than any other reportedcompound. This improved binding is an advantage for treating cancerstherapeutically.

Example 4 Activity of Compounds a, II-19, and Obatoclax in Killing HumanTumor Cell Lines

This example anticipates the activity of the compounds A, and II-19, andderivatives, in killing certain human tumor-derived cell lines grown inculture. Leukemia and myeloid cells were used to assess cell tumorkilling activity of the compounds A, and II-19, and certain derivatives.

Materials and Methods

Cell Culture

The lymphoid derived cell lines DHL-6 and DHL-10 were obtained fromAnthony Letai of the Dana Farber Cancer Research Institute, Boston,Mass. The myeloid derived cell line NCI-H929 was obtained from theNIH/NCI cell repository. AML-3 cells were obtained from Dr. MichaelAndreef (MD Anderson Cancer Center, Houston, Tex.). Cells were grown inRPMI 1640 medium (GIBCO-BRL) supplemented with Pen-strep-glutamine(Gibco 10378) and 5% fetal bovine serum.

EC₅₀ Assays

Cells were expanded in tissue culture in appropriate media and thensub-cultured into 96-well plates at a seeding density of 20,000-40,000cells per well, and cells were treated with one or more compounds thatwere titrated into appropriate medium with FCS. Cell lines were treatedwith compound A at concentrations ranging from 25 μM to 25 nM. TheBH3-mimetic obatoclax (Nguyen M et al. (2007), Proc. Natl. Acad. Sci.USA 104:19512-19517) was also tested against the SUDHL-6, SUDHL-10, andNCI-H929 cell lines. Cells were treated for 48 hours and scored forviability using the MTS assay (Promega). Growth inhibition was indicatedas a percentage of control cell growth. Growth was determined bymeasuring the A₅₇₀ (control cells)−A₅₇₀ (treated cells)/A₅₇₀ (controlcells). EC₅₀ values were calculated using Graphpad Prizm software.

Results

Cell killing by compound A is shown in FIG. 3. After 48 hours cells wereassessed for viability using the MTS assay as described above. Y axisrepresents survival as % untreated control. The X-axis represents theconcentration of the compound in log scale. Data represents mean of 3separate wells for each condition. In the multiple myeloma cell lineNCI-H929 the EC₅₀ for compound A was 1 μM for compound A (FIG. 3) and 8μM for compound II-19 (FIG. 4 a). Compound II-19 also showed activityagainst the lymphoid cell line SUDHL-6 (EC₅₀=3 μM, FIG. 4 c) and theacute myelogenous leukemia cell line AML-3 (EC₅₀=10 μM, FIG. 4 b). Noactivity was observed in the cell line SUDHL-10 (EC₅₀=>20 μM, FIG. 4 d).This cell line is devoid of Bax and Bak activity, and is therefore notresponsive to Bcl-2 family inhibition. The lack of observable activityin this assay for compound II-19 is consistent with Mcl-1 specificinhibition and suggests that this compound is not acting off-target(e.g. killing via necrotic pathways or inhibiting cell proliferation).The BH3-mimetic obatoclax (Nguyen M et al. (2007), Proc. Natl. Acad.Sci. USA 104:19512-19517) was also tested against the SUDHL-6, SUDHL-10,and NCI-H929 cell lines and showed EC₅₀ values of 2.5 uM, 0.8 uM, and1.8 uM, respectively (see FIG. 4 e for data in SUDHL-10 and NCI-H929cell lines). The activity in the BAX/BAK deficient cell line SUDHL-10indicates the compound kills cells at least in part by off-targetactivity not involving the BCL2 pathway—similar studies have beenpreviously reported (Vogler, M. et al. (2009), Cell Death andDifferentiation 16:1030-1039). This is in contrast to the cell killingprofile of compound II-19 which is disclosed herein.

These data indicate that both compounds are effective at killing tumorcells in culture and are anti-lymphoid and anti-myeloid tumor compounds.

Conclusions

Lymphoid and myeloid cells that have elevated expression of Mcl-1 tendto be resistant to certain chemotherapies. This includes multiplemyeloma (MM) (Zhang B et al. (2002), Blood 99:1885-1893), non-Hodgkin'slymphomas (Cho-Vega J H et. at (2004) Hum. Pathol. 35(9):1095-100) andchronic lymphocytic leukemia (CLL) (Michels J, et al. (2004), Oncogene23:4818-4827) cells. As shown in Example 2 above, compound A andcompound II-19 target Mcl-1. This example sets out to show that elevatedMcl-1 would cause hypersensitivity to compound A and II-19 under certainconditions. It is more likely that hypersensitivity will occur when theBH3-only protein, Bim, Puma or Noxa are also elevated. Both of theseproteins have BH3-mediated binding to Mcl-1. Therefore these cells willbe more sensitive to whichever of the BH3 mimic compound A or II-19 thathave activity in disrupting Bim, Puma or Noxa BH3 mediated binding toMcl-1.

Compounds described herein are effective in killing tumor cells thathave elevated Mcl-1, and those that have elevated Mcl-1 and elevatedBH3-only protein Noxa and/or Puma. The BH3 mimic compounds describedherein are effective at treating chemo-resistant MM, CLL, NHL, AML, andALL cells that display elevated Mcl-1. Compounds described herein arealso effective as second line therapy in patients treated withproteosome inhibitors such as Bortezomib (Velcade®) who display elevatedMcl-1 with or without elevated Bim, Puma, or elevated Noxa.

Example 5 In Situ Mitochondrial Assay for Compound a

The on-target activity of compound A was validated. Changes inmitochondrial membrane potential were observed utilizing thepotentiometric dye JC-1 (Invitrogen, Carlsbad, Calif.) a mitochondrialdye that loses its 590 emission signal when the outer membrane of themitochondria losses its membrane potential. (Deng et al. (2007) CancerCell. 12(2):171-85).

The selective response of mitochondria in semi-permeabilized cell linesto the compounds was observed. The assay was performed as described in(Deng et al. (2007) Cancer Cell. 12(2):171-85).

Materials and Methods

Suspension cells were grown in RPMI and re-suspended to a 4× assayconcentration of 2×10⁴/ml in assay buffer. Assay buffer; 300 mMTrehalose, 10 mM HEPES-KOH pH 7.7, 80 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1%BSA, 5 mM Succinate. Cells are mixed with JC1 and allowed to label for10 minutes at 37° C. and then test compounds or control peptides areadded in a final reaction. Final assay conditions are 2.5×10⁴ to 1×10⁵cells/well, 0.005% digitonin, 5 mM BME, 40 μM control peptide (incontrol wells) or 4 to 0.25 μM test compound (in test wells), 30 μLtotal volume per well in Greiner 384 flat bottomed well white plates.Plates are read in Biotek, Fluorescence plate reader; 530 nm excitation;590 nm emission. Loss of JC-1 signal emission wave length (590 nm) isrepresented as a percent of the non-treated (DMSO control).

Results

Response of mitochondria in semi-permeablized lymphoid cell line SUDHL-6to compound A is shown in FIG. 5. This cell line has functioning Bax andBax pro-apoptotic protein and is expected to respond to BH3 mediatedactivation. Loss of JC-1 signal emission wave length (590 nm) isrepresented as a percent of the non-treated (DMSO control). Lack ofresponse to compound A in mitochondria from Bax/Bak deficient cell lineSUDHL-10 is shown in FIG. 6. This cell line does not have functioningBax and Bax pro-apoptotic protein and is not expected to respond to BH3mediated activation. These data indicate direct activity of the compoundA (ranging from 4 μM to 0.25 μM) on Mcl-1 primed mitochondria (FIG. 5),and that the Bax/Bak activity is required for activity (FIG. 6). This isconsistent with the compounds acting through the mitochondrial apoptosispathway.

Example 6 In Situ Mitochondrial Assay for Compound II-19

The on-target activity of compound II-19 was validated. Changes inmitochondrial integrity were observed utilizing anti-cytochrome cconjugated to Alexa488 (BD). When the mitochondria are intact, theyretain cytochrome c and have bright, punctate staining with the antibodywhereas cells with compromised mitochondrial integrity will losecytochrome c and will not stain with the antibody. This can be observedby microscopy as well as measured by a shift in fluorescence on the FL1channel of a flow cytometer.

The selective response of mitochondria in semi-permeabilized cell linesto the compounds was observed. The assay was adapted from (Campos et al.(2006) Cytometry Part A. 69(A):515-523).

Materials and Methods

Suspension cell lines SUDHL10 and SUDHL6 were grown in RPMI, washed oncein 1×PBS and re-suspended at a concentration of 2e6/ml in assay bufferwith 0.0025% Digitonin. Assay buffer; 300 mM Trehalose, 10 mM HEPES-KOHpH 7.7, 80 mM KCl, 1 mM EGTA, 1 mM EDTA, 0.1% BSA, 5 mM Succinate. Cellsare incubated with test and control compounds at 10⁶ cells/treatment for1 hour at room temperature. Samples are fixed with 4% formaldehyde inPBS for 20 minutes, washed once in PBS, and blocked with 2% FBS/0.5%TritonX-100 in PBS. Samples are re-suspended in blocking buffer with1:250 anti-cytochrome c conjugated to Alexa488 (BD Cat#56028) for 1 hourat 4° C., washed once with blocking buffer and re-suspended in 200 ulPBS. Cytochrome c loss was measured by microscopy. At least 100 cellsper treatment were counted and scored as positive for cytochrome c lossif they lacked staining. In both methods DMSO was calculated as 0%cytochrome c loss and the Bim response for DHL6 was used to determine100% cytochrome c loss.

Results

These data indicate direct activity of the compound II-19 at 10 uM onprimed mitochondria, and that the Bax/Bak activity is required foractivity (compare activity in Bax/Bak-functional cell line (DHL-6) tolack of activity in Bax/Bak-deficient cell line (DHL-10) (FIG. 7). Thisis consistent with the activity profile of these compounds in cellstudies, and indicates that the compounds exert their biological effectthrough the mitochondrial apoptosis pathway.

Example 7

This example demonstrates the efficacy of compounds described herein inpotentiating the tumor cell killing activity of Bortezomib (Velcade®).

Proteosome inhibitors such as Bortezomib induce apoptosis and have beenrecognized as a class of anti-tumor therapeutics (Adams (2004) CancerCell 5: 417-421). Bortezomib (Velcade®) has been approved to treatA-myeloid leukemias and is in phase 3 trials for treatment of solidtumors. Bortezomib is known to dysregulate proteosome-mediatedmaintenance of Mcl-1 levels in the cell. Accumulated Mcl-1 inBortezomib-treated cells has been shown to reduce cell killing andpromote tumorigenesis, while reduction of Mcl-1 in cells enhances theeffectiveness of Bortezomib in inducing apoptosis (Nencioni et al.(2005) Blood 105: 3255-62). Further, Bortezomib has been shown to causeelevated expression of the BH3-only protein Noxa (Qin et al. (2005)Cancer Res. 65(14): 6282-93). The combination of elevated expression ofMcl-1 and Noxa is likely to lead to a cell state previously described asBH3 “sensitization” (Letai et al. (2002) Cancer Cell. 2(3): 183-92.) andmake these cells particularly responsive to Mcl-1 specific inhibition.

Materials and Methods

The experiments are performed in Jurkat cells or in primary A-myeloidleukemia cells. Cells are treated with Bortezomib alone or incombination with compound A, or II-19, or derivatives and GI₅₀ valuesare determined using the MTS assay as described in Example 4.

Jurkat cells are obtained from the American Type Culture Collection(ATCC) Manassas, Va. Primary AML cells are described (Milella et al.(2002) Blood 99(9): 3461-64) and can be obtained from Dr. MichaelAndreeff, M.D. Anderson Cancer Center, Houston, Tex. Mcl-1 rabbitpolyclonal anti-Human Mcl-1 IgG is available from Cell SignalingTechnologies (Beverly Mass.). RPMI 1640 medium is available fromGIBCO-BRL (Carlsbad, Calif.).

MTS cell viability reagents are available from Promega, (Madison, Wis.).Bortezomib (Velcade®, Millennium Pharmaceutical, Cambridge, Mass.) isavailable with prescription from any pharmacy.

Cells are to be planted in 96-well plates at 2×10⁴ cells/well andincubated in 200 μl RPMI with 10% fetal calf serum with antibiotics for48 hours. Bortezomib is titrated in a 2 fold serial dilution that rangesfrom 5 to 320 ng/ml (5, 10, 20, 40, 80, 160, 320 ng/ml). Treated cellsare allowed to incubate for 48 hours. Treated cells are then assessedfor viability using the MTS assay as described in Example 4. The GI₅₀ isdetermined.

Combination treatment of Bortezomib and compounds of Formula Ia′, Ib′,Ic′, or II′ is performed after the GI₅₀ of Bortezomib is established.Cells are treated with three concentrations of Bortezomib, the GI₅₀ and2.5 and 5 fold lower concentrations. To these treated cells, thecompounds of Formula Ia′, Ib′, Ic′, or II′ is added simultaneously inconcentrations of 10, 5, 2.5, 1.25, 0.67, 0.34, 0.17, 0.08, 0.04 and0.02 μM. Combination treated cells and cells treated with eitherBortezomib alone or the BH3 mimic compounds of Formula Ia′, Ib′, Ic′, orII′, alone are assessed for viability following 24, 48, and 72 hoursusing the MTS assay protocol described in Example 4.

Further analysis of cell death is conducted using fluorescence-activatedcell sorting (FACS) analysis of annexin V positive staining with thevital dye propidium iodide by standard methods and as described inWilkins et al. (2002) Cytometry 48(1): 14-9. Determination of enhancedkilling with Bortezomib is correlated with expression levels of Mcl-1 asdetermined by western blotting of Bortezomib treated and non-treatedcells, as well as combinations of treated cell lysates with anti-Mcl-1antibodies (Cell Signaling Technologies, Beverly, Mass.).

Results

Treatment of cells with the Bortezomib (Velcade®) has been shown tocause elevated Mcl-1 as well as elevated Noxa in lymphoid cells(Perez-Galan et al. (Sep. 15, 2005) Blood 107:257-264; Qin et al. (2005)Cancer Res. 15: 65(14): 6282-93). This combination of elevated Mcl-1with elevated Noxa leads to the condition described by Letai as “primed”to respond to an Mcl-1 inhibitor.

The compound or compounds among the compounds of Formula Ia′, Ib′, Ic′,or II′, that best inhibit Mcl-1 will be most effective in synergizingwith Bortezomib and increasing its effective range.

Because the chemotherapeutic compounds taxol and doxorubicin mediatecell death through the activation of the tumor suppressor p53, andbecause Noxa is downstream of p53, it is anticipated that thesetreatments will also cause Mcl-1 over-expressing cells to become“primed” for Noxa mediated death when the Mcl-1/Noxa complex isdisrupted, as would be the case in treating with a Mcl-1 specific BH3mimic compound. Therefore it is anticipated that the compound(s) ofFormula Ia, Ib, Ic, or II that best inhibit Mcl-1 will be most effectivein synergizing with taxol or doxorubicin and increasing the effectiverange of these compounds in killing tumor cells in vitro. It isanticipated that this efficacy will transfer to killing of tumor cell invivo in animal models for hematological malignancies and to therapeuticvalue in treating hematological malignancies in humans.

Example 8 In Vivo Activity of Compound a in a Mouse/Human XenograftModel for Lymphoma

Compound A was tested in SCID mice at SRI International, Menlo Park,Calif. A dose escalation study determined that that the high dose of 60mg/kg was well-tolerated and could be safely used for the xenograftstudy for all 3 test articles.

The T Lymphoblastic Leukemic Cell Line CCRF-CEM was purchased fromAmerican Type Culture Consortium (ATCC). These cells were maintained inRPMI-1640 medium supplemented with 10% of fetal bovine serum, 2 mMglutamine, and 1 mM sodium pyruvate. The cells were cultured at 37° C.in 95% air/5% CO₂ and 100% humidity. Medium in the culture was changedevery 48 h and cells were passaged weekly.

For cell implantation to mice, cells were harvested by centrifugation.Cell pellets were resuspended in PBS and counted using a hemacytometerand Trypan Blue dye to measure the number of viable cells in thesuspension. The harvested cells were washed once with PBS andresuspended in serum-free medium at a density of 1×10⁷ cells/100 μl.

Study Design

For this xenograft study, animals were divided into 4 groups of 10 miceeach. Groups were treated with test compounds by IP injection at 60mg/kg with or vehicle control.

Experimental Design for the CCRF-CEM Xenograft Study

Group # of Mice Treatment Route Regimen 1 10 Vehicle* control I.P.5x/week 2 10 Compound A, 60 mg/kg I.P. 5x/week *Vehicle =DMSO/Cremophor/water (10:18:72)Cell Implantation

Mice were inoculated with the CCRF-CEM cells (10,000,000 cells) were bytail vein injection. The cell suspensions had the tendency to formclumps. Therefore, during cell inoculation, the cell suspensions had tobe mixed well before drawing into the syringe. Gauge 28 needles wereused for the injection.

Assay Procedure

Test articles were dissolved in a mixture of DMSO/Cremophor/water(10:18:72) and administered to the animals in 100 μl aliquots 5× perweek beginning 7 days after cells were transplanted into mice. Controldosage was performed under the same schedule.

Throughout the entire study, clinical observations were conducted dailyfor signs of leukemia development, including lethargy, ruffled fur, lackof responsiveness to stimuli, weight loss, and becoming moribund. On Day21 after cell inoculation, blood samples were drawn from all groups. Thewhite blood cells of all the blood samples were measured with a NucleoCounter (ChemMetec, Denmark). The clinical observations were continueduntil animals either died spontaneously or sacrificed when they becamemoribund.

Compound Pharmacokinetics

Pharmacokinetic studies were conducted on compound A to assess plasmaclearance and distribution in the mouse. Compound A was administeredi.p. (as in the xenograft study) at 10 mg/kg. Compound A exhibited goodstability in the mouse with plasma half-lives of 2.1 h Consistent withthe excellent microsome stability, the compound exhibited low plasmaclearance values 0.08 L/h/kg (compound A). These data indicate that druglevels in the xenograft studies were maintained above the IC₅₀ of thecompound throughout the dosing regime.

White Blood Cell Counts in Treated and Untreated Mice

After 14 days of treatment, blood samples were taken from all the micevia orbital bleeding while mice were anesthetized using isofluraneinhalation. Approximately 100 μl of blood was taken from each mouse. Thenumber of white blood cells was then counted by standard methods.

Results

As shown in FIG. 8, the white blood cells of mice in groups treated withcompound A were significantly reduced when compared with the vehiclecontrol group with P<0.02 (Students' “t” test).

During the first 20 days after the inoculation of 1×10⁷ CCRF-CEM cellsvia i.v. injection through the tail vein, none of the mice showedclinical signs of leukemia development. However, by day 22, 5 mice inthe control group died. The survival outcome is shown in FIG. 9.Compound A was statistically significant in effecting survival outcome.

Conclusion

This study demonstrated that compound A has biological activity andlowered white blood cell counts and ultimately effected survival outcomesignificantly when compared with vehicle control as shown in FIG. 9. Thereduction of white blood cells was statistically significant for testarticle Compound A with P<0.02 (Students' “t” test) (FIG. 8). Thesurvival outcome of mice treated with compound A was significantlyimproved over the non-treated animals. On Day 21, 3 mice in the vehiclecontrol began to develop ruffled fur and became less active. On Day 22,all 5 mice died. Beginning on Day 22, mice in each group began to showabnormal clinical signs. Animals either died spontaneously with time orbecame moribund. The moribund mice were sacrificed humanely in a timelymanner. All mice in the vehicle control group died by Day 31.60% of themice in group treated with compound A were still alive on Day 31.

Pharmacokinetic studies were conducted on compound A to assess plasmaclearance and distribution in the mouse. Compounds were administeredi.p. (as in the xenograft study) at 10 mg/kg. Both compounds exhibitgood stability in the mouse with plasma half-live of 2.1 h.). Consistentwith the excellent microsome stability, compound A exhibited low plasmaclearance values 0.08 L/h/kg. These data indicate that drug levels inthe xenograft studies were maintained above the IC₅₀ of the compoundthroughout the dosing regime.

Oral formulations are made and tested using the Pharmatek Lab orAtories, Inc. (San Diego Calif.) Hot Rod Chemistry formulation screeningkit. This kit is designed to find the correct formulation to solubilizecompounds that normally have poor solubility characteristics. The kitcontains eight formulations to test. Accordingly, the formulation, whichimparts the best solubility, bioavailability, and stability for a leadcompound is identified.

Example 9 Activity of Compound II-20 in the Fluorescence PolarizationAssay Measuring Mcl-1 and Bcl-xL Inhibition

The compound II-20 was assessed for ability to inhibit binding of theBH3 peptide of Bim to Mcl-1 and Bcl-xL. The activity for blocking BimBH3 binding to both Bcl-xL and Mcl-1 fusion proteins are measured andcompared as in Example 2.

Materials and Methods

Experiments are performed using the fluorescence polarization assay asdescribed in Example 2. Either GST-Bcl-xL or GST-Mcl-1 fusion proteinswere titrated and maximum mp shift indicate binding concentrations fordrug titration studies, as described in Example 2. Compound II-20, wastitrated from 10 μM to 0.1 nM in 2-fold serial dilution into thepeptide/protein solution. The IC₅₀ for blocking each BH3 peptide fromMcl-1 are assessed using FP, as described in Example 2.

Results

Compound II-20 with a drug concentration that provokes a responsehalfway between baseline and maximum (IC₅₀) of 27 nM for Mcl-1 and 122nM for Bcl-xL. Compound II-20 has approximately a 5-fold preference forinhibition of Mcl-1 vs. Bcl-xL as seen in FIG. 10.

Example 10 Activity of Compound II-20 in Cell Viability Assays

The ability of compound II-20 to reduce cell viability of cancer celllines was tested in SUDHL-6 and SUDHL-10 according to Example 4.Compound II-20 showed activity against the SUDHL-6 cell line (EC₅₀=9.7μM) but not the SUDHL-10 cell line (EC₅₀>25 μM), indicating that Bax andBak are required for cell killing activity, a mode of action consistentwith Mcl-1 specific inhibition.

Example 11 Pharmacokinetics of Compound II-19 Administered i.p. in Mice

Pharmacokinetic studies were conducted on compound II-19 to assessplasma clearance and distribution in male ICR mice. Compounds wereadministered i.p. (as in the xenograft study, Example 8) at 10 mg/kg.Blood samples were collected at various time points (pre-injection, 5min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 24 h), and centrifuged toremove red blood cells. The plasma layer was recovered, and extractedwith acetonitrile (equal volume) containing 1% formic acid. Plasmalevels of drug were quantified by LC-MS (C18 column eluted with lineargradient from 0% acetonitrile, 1% formic acid to 100% acetonitrile, 1%formic acid) against a set of standards prepared by diluting a knownquantity of II-19 into mouse plasma.

Results

The compound exhibits good stability in the mouse with plasmahalf-live >20 h (FIG. 11). The results indicate that significant levelsof circulating drug can be achieved in the mouse and that these levelscan be sustained for >24 h. Significantly, the levels achieved are closeto or in excess of the EC₅₀ observed in several cell lines (NCI H929,AML-3) for inhibition of multiple myeloma cell viability indicating thatthe levels of drug are potentially high enough to have a therapeuticeffect against myeloma cancer cells in vivo.

Example 12 Activity of Compound II-19 in a Mouse Human Myeloma XenograftModel

Efficacy Study

Cell Culture: Tumor cell line NCI H929 was cultured according to ATCCrecommendations in RPMI8226 media containing 10% fetal bovine serum andpen-strep. On the day of implantation, cells were washed 1× withphosphate buffered saline (PBS) and harvested via centrifugation. Cellswere pelleted (5 min at 1000 rpm, RT) then resuspended in serum-freematrigel and kept on ice until implantation.

Tumor Cell Implantation: The cell line was implanted via subcutaneousinjection on the right rear flank at a volume of 200 μl containing3×10^7 cells per mouse; the injection site was cleaned using alcoholbefore and after injection to prevent any infection.

Tumor Assessment: Beginning 1 week post cell implantation all animalswere weighed and tagged and tumors were measured and body weights taken3 times weekly (length×width×height) on Monday, Wednesday, and Fridayfor the duration of study. Once an average tumor volume of 100-200 mm³was reached, animals were randomized and placed into 4 treatment groupsof a minimum of 10 mice per group based on tumor size. Randomization wasdetermined by tumor volume (length×width×height)/0.52 for all of themice and average volume was calculated using a average function in Excel(Microsoft Professional).

Treatment: The study involved 4 groups of 10 animals per group. Animalsin all dose groups were weighed on days of tumor measurement. All doseswere administered i.p. at 10 mL/kg. The Bortezomib (1 mg/kg) group wasonce every 3 days for 15 days. The mice receiving vehicle and compoundII-19 were dosed every second day. Compound II-19 was formulated in 1%DMSO, 10 mM Tris pH 8, 30% hydroxypropyl-beta-cyclodextrin; this vehiclewas used as the “vehicle” control. Bortezomib was formulated in 30%propylene glycol, 5% Tween 80, 3.3% dextrose in water, pH 4, 1% DMSO.

Results

Animals treated with both the 1 mg/kg eod and 3 mg/kg eod doses ofcompound II-19 exhibited a lower tumor burden than animals treated withvehicle (FIG. 12). At the end of the study, vehicle treated animals hadon average a tumor size of 2300 mm³ which was reduced in the 1 mg/kg and3 mg/kg compound II-19 treated animals to 1740 mm³ and 1600 mm³,respectively. Tumor volume in the bortezomib treated animals was alsoreduced compared to vehicle with an average volume of 1180 mm³. Theresults indicate that compound II-19 exhibits anti-tumor activity invivo against multiple myeloma cancer cells that inhibit tumor growth anddisease progression.

Example 13 Summary Structure-Activity Results for Derivatives ofCompound A

In addition to compound A (see example 2), compound II-19 (see Example3) and compound II-20 (see Example 9), compounds Ic-1, Ic-6, Ic-7, Ic-8,Ic-17, and Ic-18 were tested for their ability to inhibit MCL1 by thefluorescent polarization assay described in Example 2 and Example 3.These compounds inhibited MCL1 with IC₅₀ values of <50 μm.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

What is claimed is:
 1. A compound selected from dimethyl4,4′-diformyl-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylateand dimethyl1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-[2,2′-biphenazine]-9,9′-dicarboxylate.2. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 3. A compound, wherein the compoundis dimethyl4,4′-diformyl-1,1′,3,3′,6,6′-hexahydroxy-[2,2′-biphenazine]-9,9′-dicarboxylate.4. A pharmaceutical composition comprising the compound of claim 3 and apharmaceutically acceptable carrier.
 5. A compound, wherein the compoundis dimethyl1,1′,3,3′,6,6′-hexahydroxy-4,4′-bis(hydroxymethyl)-[2,2′-biphenazine]-9,9′-dicarboxylate.6. A pharmaceutical composition comprising the compound of claim 5 and apharmaceutically acceptable carrier.